Immunoglobulins and variants directed against pathogenic microbes

ABSTRACT

Anti-SpA murine, chimeric and humanized monoclonal antibodies, and variant antibodies having a heavy chain with at least one amino acid substitution are provided. Such antibodies may be used to prevent or treat microbial infections.

PRIORITY CLAIM

This application is a continuation in part of U.S. patent applicationSer. No. 14/312,585, filed Jun. 23, 2014 and now pending; which is acontinuation of International Patent Application No. PCT/US2012/071556,filed Dec. 23, 2012 and now expired; which claims priority to U.S.Provisional Application No. 61/580,194, filed Dec. 23, 2011 and nowexpired; the subject matter of all of which is hereby incorporated byreference as if fully set forth herein.

BACKGROUND

Pathogenic microbes, such as gram-positive cocci, can produce an arrayof secreted or cell surface-associated virulence factors, which arecapable of interfering with the host immune responses. A number of suchvirulence factors are binding proteins, which contain one or moredomains that bind to host immunoglobulins. Such Immunoglobulin bindingvirulence factors which bind to the heavy chain constant region of theimmunoglobulin are referred to at Immunoglobulin Binding Proteins(IgBPs). A subset of Immunoglobulin Binding Proteins, which interactwith the Fc region of immunoglobulins, are referred to as Fc Bindingproteins (FcBPs). This non-immune binding of immunoglobulins by IgBPsinvolves regions of the immunoglobulin outside of the antigen-antibodycombining site. Such non-immune binding of host immunoglobulins bymicrobial virulence factors is thought to subvert the host antimicrobialimmune response. Functionally, this can occurs through immuno-shieldingby coating of the bacteria with Fc bound antibody, by blockingimmunoglobulin Fc-mediated effector functions such as complementactivation or Fc-receptor mediated binding to effector cells, or byexpression of superantigens which interact with immune cell surfacereceptors.

In the case of Staphylococcus aureus (S. aureus), a number ofimmunoglobulin binding proteins are expressed, including Protein A(SpA), Sbi, SSL7 and SSL10.

There is some evidence that it is possible to generate an antibodyresponse to highly purified surface components of S. aureus such ascapsular polysaccharide, the collagen-binding protein Cna and thefibrinogen-binding protein ClfA. This has led to the discovery andclinical testing of a number of antibodies based therapies directedagainst such S. aureus antigens.

Despite promising preclinical activity, clinical trials of such agentshave been met with little success. Infants with very low birth weights(<1500 g; <32 weeks gestation) are at a particular risk for nosocomialbacterial infection, as they have not benefited from trans-placentaltransfer of maternal antibodies. Many of these infections are caused byS. aureus. Altastaph® is a vaccine-induced hyperimmune polyclonalantibody with specificity for S. aureus serotype 5 and 8, developed byNabi Biopharmaceuticals (US20060153857 A1). In spite of reaching targetserum antibody levels, no decrease in S. aureus infection rates wasobserved in treatment groups in two clinical trials (Rupp et al., 2007;DeJonge et al., 2007). A second anti-S. aureus human immune sera,INH-A21 (Veronate®) was prepared by first screening donors for hightitres against MSCRAMM (microbial surface components recognizingadhesion matrix molecules), (Inhibitex—U.S. Pat. No. 6,692,739).Although Phase II trials appeared promising at the highest antibodydose, Phase III of the trial did not observe any effect of antibodytreatment in reducing the frequency of S. aureus infection.

Additional antibodies or antibody derived molecules which have beenunder development include Aurograb, an antibody that targets theimmunodominant ABC transporter in MRSA (Weems et al., 2006), which wasdesigned to blocks the multi-drug efflux pump, allowing antibiotics toretain effectivity; tefibazumab (Aurexis®) (U.S. Pat. No. 6,979,446),which targets Clumping Factor A (ClfA) and pagibaximab (BYSX-A110,US20080019976 A1), a chimeric antibody which binds lipoteichoic acid(LTA) present in the membrane of gram-positive bacteria. ElusysTheraputics has also attempted to developed a bispecific heteropolymerantibody by cross-linking an antibody directed against SpA with a secondantibody the recognizes the CR1 receptors (WO 2008/140487 A2).

Currently, none of the approaches described above have shown significantactivity in clinical trials. The development of new antibody basedagents which overcome microbial immune evasion for the treatment orprevention of microbial infections, including S. aureus, is an importantgoal that would be of great clinical benefit.

SUMMARY

The embodiments described herein provide for anti-microbial variantantibodies, which have attenuated non-immune binding (binding toresidues outside of the antigen-antibody combining site) to one or moremicrobial immunoglobulin binding proteins (IgBPs).

According to the embodiments described herein, the disclosure providesanti-microbial monoclonal antibodies. In one embodiment, ananti-microbial variant antibody is provided that includes animmunoglobulin heavy chain (e.g., an IgG heavy chain) that differs fromthat of its unmodified parent anti-microbial antibody by at least oneamino acid substitution, wherein the variant immunoglobulin heavy chainhas attenuated non-immune binding to one or more microbial virulencefactors as compared to that of the unmodified parent antibody. In oneaspect, the variant anti-microbial IgG antibody includes a variant heavychain, in which at least one amino acid from the IgG heavy chainconstant region is substituted with another amino acid which isdifferent from that present in the parent antibody.

In some embodiments, the monoclonal antibody is a chimeric, humanized ofhuman anti-microbial IgG variant antibody, in which at least one aminoacid from the IgG heavy chain constant region, is substituted withanother amino acid which is different from that present in the parentantibody. Such variant anti-microbial antibodies have attenuated heavychain constant region binding to one or more microbial IgBPs or IgBPdomains expressed by the target microbe.

The variant immunoglobulin IgG heavy chain constant regions describedherein can be combined with immunoglobulin variable heavy and lightchain regions which bind antigens produced by microbes that express oneor more microbial IgBP.

In some embodiments, the variable domain of the antibody binds to amicrobial protein that is a microbial immunoglobulin binding protein,and the heavy chain constant region of the antibody is a variant whichhas attenuated binding to one or more microbial IgBPs or IgBP domainsexpressed by the target microbe.

In other embodiments, the variable domain of the antibody binds to amicrobial protein that is not an microbial immunoglobulin bindingprotein, and the heavy chain constant region of the antibody is avariant which has attenuated binding to one or more microbial IgBPs orIgBP domains expressed by the target microbe.

The anti-microbial heavy chain constant region variant IgGimmunoglobulins claimed herein have enhanced antimicrobial activityrelative to their parental antibodies. For example, in the case of S.aureus, an important human pathogen for which there is an urgent unmettherapeutic need, a number of IgBPs can be expressed, including SpA,Sbi, SSL7 and SSL10.

In some embodiments the target microbe is S. aureus. Heavy chainconstant region variant IgG immunoglobulins are described, which haveattenuated binding to one or more S. aureus IgBPs due to theintroduction of one or more amino acid substitutions in the heavy chainconstant region domain relative to the parental IgG.

In some embodiments in which the target microbe is S. aureus, such heavychain constant region variant IgG polypeptide sequences are combinedwith immunoglobulin heavy chain variable polypeptide sequences and lightchains polypeptide sequences, which bind one or more cell surface orsecreted S. aureus antigen.

In some embodiments, the S. aureus antigen recognized by the variabledomain of variant antibodies are cell surface or secreted antigensselected from the list which includes but is not limited to: ClfA, ClfB,Cna, Eap, Ebh, EbpS, FnBPA, FnBPB, IsaA, IsaB, IsdA, IsdB, IsdH, SasB,SasC, SasD, SasF, SasG, SasH, SasK, SdrC, SdrD, SdrE, Spa, SraP, Coa,Ecb, Efb, Emp, EsaC, EsxA, EssC, FLIPr, FLIPr like, Sbi, SCIN-B, SCIN-C,VWbp, SpA, LTA, CP5, CP8, PNAG, dPNAG, alpha toxin, CHIPS, PVLleukocidin, α, β and γ-hemolysins, SAK, Sea, Sep, Seb, Epa, Efb, SCIN,Exfoliatins ETB and ETA, Staphylococcal Enterotoxins SEA, SEB, SECn,SED, SEG, SHE, and SEI, Toxic-shock syndrome toxin TSST-1, Alpha Toxin,Beta toxin, Delta toxin.

In some embodiments, the antigen recognized by the variable domain ofthe antibody or its heavy chain constant region variants is S. aureusSpA. In such embodiments, the microbial antigen recognized by thevariable domain of the variant IgG antibody is an epitope found in oneor more of the repeat homology IgBP domains of S. aureus SpA (referredto as SpA domains E, D, A, B, and C).

In some embodiments, the antigen recognized by the variable domain ofthe antibody or its heavy chain constant region variants is S. aureusSbi. In such embodiments, the antigen epitope recognized by the variabledomain of the antibody or its variants is located in one or more of theSbi IgBP binding domains I and II.

In some embodiments, the antigen epitope recognized by the variabledomain of the antibody or its heavy chain constant region variants isfound in two or more of the repeat IgBP homology domains of SpA or Sbi,selected from the list SpA domains E, D, A, B, and C, and Sbi domains Iand II.

In some embodiments, the antigen epitope recognized by the variabledomain of the antibody or its heavy chain constant region variants isfound in one more of the repeat IgBP homology domains of both SpA andSbi, selected from the list SpA domains E, D, A, B, and C, and Sbidomains I and II.

Described herein are methods of producing monoclonal antibodies thatrecognize SpA and/or Sbi, methods for selecting antibodies that crossreact with multiple SpA IgBP domains (selected from SpA domains E, D, A,B, and C) and/or Sbi (selected from Sbi domains I and II), methods ofselecting antibodies that cross react with one or more SpA IgG bindingdomains and Sbi domains I and/or II, methods of assaying for antigenbinding to SpA or Sbi using variant IgG1 antibodies, having one or moreamino acid substitutions in the heavy chain constant region whichprevent heavy chain constant region binding to SpA, Sbi or SSL10. Insome aspects, the variant Fc domain used for antibody selection is ofhuman isotype IgG1 having one or more of the following amino acidsubstitutions: a His to Arg substitution at position 435, a Tyr to Phesubstitution at position 436 and a Arg to Gln at position 274. In oneaspect, the variant Fc domain used for antibody selection is of humanisotype IgG1 and has a His to Arg substitution at position 435, a Tyr toPhe substitution at position 436 and a Arg to Gln at position 274. Inanother aspect, the variant Fc domain used for antibody selection is ofhuman isotype IgG1 and has a His to Arg substitution at position 435.The uses of such Fc variants are important so as to differentiateantigen specific binding of the antibody from Fc mediated binding to Sbiand or SpA (positions refer to EU numbering).

In an additional embodiment, modification of human or humanized VH3family derived anti-S. aureus IgG variable heavy domain residues areclaimed which abrogate superantigen type binding of SpA to anti S.aureus immunoglobulins or their heavy chain constant region variants.

In an additional embodiment, the antigen recognized by the variabledomain of the claimed heavy chain constant region variantimmunoglobulins is S. aureus Clumping factor A (ClfA).

In additional embodiments, heavy chain constant region variant anti-S.aureus antibodies are provided in which the human, humanized, orchimeric variable domain, or variable domain CDRs of the antibody arederived from an anti-S. aureus antibodies selected from the list:Pagibaximab (a chimeric anti-LTA antibody; Biosynexus/Medimmune),Tefibazumab (a humanized IgG1 anti-ClfA; Aurexis, Inhibitex/BMS), CS-D7(human anti-IsdB IgG1, Merck), Aurograb (scFv fragment anti ABCtransporter; NeuTec/Novartis), anti-Alpha toxin (Medimmune patentapplication WO/2012/109285).

In other embodiments, affinity matured heavy chain constant regionvariant anti-S. aureus antibodies are provided in which human,humanized, or chimeric variable domain of the antibody are derived froman anti-S. aureus antibodies selected from the list including, but notlimited to: Pagibaximab (a chimeric anti-LTA; Biosynexus/Medimmune),Tefibazumab (a humanized IgG1 anti-ClfA, Inhibitex/BMS), CS-D7 (ahumanized anti-IsdB IgG1, Merck), Aurograb (an scFv fragment anti-ABCtransporter; NeuTec/Novartis), anti-Alpha toxin (Medimmune patentapplication WO/2012/109285). Such claimed affinity matured Heavy chainconstant region variant antibodies have at least one amino acidsubstitution, deletion or insertion relative to the parental heavy orlight chain variable domain sequences.

In additional embodiments, the disclosure also relates to theprophylactic or therapeutic use of such anti-microbial immunoglobulinsand their heavy chain constant region variants, and their use incombinations with additional antimicrobial chemotherapy oranti-infective agents or in combination with one or more additionalantimicrobial immunoglobulins or variant immunoglobulins.

The disclosure also relates to the prophylactic or therapeutic use ofsuch anti-microbial immunoglobulins and their heavy chain constantregion variants, and their use in combinations with additionalantimicrobial chemotherapy or anti-infective agents or in combinationwith one or more additional antimicrobial immunoglobulins or variantimmunoglobulins for use in veterinary or animal use.

The anti-microbial heavy chain constant region variants immunoglobulinsdescribed herein, which have enhanced anti-microbial activity relativeto their parental antibodies, may be used for the prophylactic ortherapeutic treatment of a number of important infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the domain organization of an IgGpolypeptide according to some embodiments.

FIG. 2 is a schematic diagram illustrates the domain organization of theSpA (SpA) polypeptide (Panel A) and shows the sequence alignment of thefive highly homologous extracellular Ig-binding domains of SpA intandem, designated domains E, D, A, B, and C (Panel B).

FIG. 3 is a schematic representation of a complex between SpA domain Dand Fab 2A2 from a human IgM. (Panel A) shows a side view of SpA domainD bound to the framework region of the Fab heavy chain. The VL domain,which is not involved in this interaction, is shown on the right. TheCDR loops as defined by Chothia and Lesk are highlighted at the top.(Panel B) shows a schematic diagram detailing the residues of SpA domainD and Fab 2A2 involved in the interaction. Kabat numbering is used forthe VH residues; domain D is numbered with the convention used for SpAdomains.

FIG. 4 is a schematic diagram illustrating the SpA (top) and Sbi(bottom) polypeptide domain organization.

FIG. 5 shows a sequence alignment of human IgG sequences IgG1, IgG2,IgG3 and IgG4 for the CH1 region, the Hinge Sequence, the CH2 sequenceand the CH3 sequence.

FIG. 6 is a table showing Human IgG1 allotypes.

FIG. 7 illustrates the allotypes of the gamma chain of human IgG3. Thepositions of amino acid substitutions in the gamma chain of IgG 1, 2 and4 are compared to IgG3 allotypes.

FIG. 8 illustrates domain B of Spa with inter-domain substitutions.Panel A shows domain B of SpA interacting with an IgG Fc domain.Inter-domain substitution positions are shown in yellow and by arrows.Panel B shows a close up of the interaction of SpA domain B with the IgGconstant region. The structure encompasses the IgG CH2-CH3 interface.Alpha Helices are shown as cylinders (within the circle). SpA Helix Iand II (Helix III is not shown), which form the binding interface withresidues within the IgG CH2-CH3 region, are shown. Amino acids of thebackbone (worm representation) and side chains which vary betweendomains B and domains E, D, A and C (FIG. 2) are shown in yellow and byarrows. Diagram made using Cn3D using and PDB ID 1FC2).

FIGS. 9A-9C are a series of diagrams showing domain D of SpA interactingwith the Fab domain of a human IgM. FIG. 9A shows Domain D of SpAinteracting with the Fab domain of a human IgM. The diagram is adaptedfrom Grille, et al., 2000 using Cn3D (PDB ID: 1DEE). Helix II and III,which form the binding interface with residues within the VH3 Fab, areshown. Amino acid backbone (worm representation) and side chains areshown. The position of amino acids which vary between domains E, D, A,B, and C (FIG. 2) are shown in yellow (inter-domain substitutions). Thecontact residues which form the binding interface are conserved amongall SpA Ig-binding domains, suggesting that each could bind in a similarmanner. FIGS. 9B and 9C show close ups of the interactions fromdifferent angles.

FIG. 10 models a Q to K substitution at amino acid 45 of SpA domain E.Amino acid K is found at the same position in all sequenced stains of S.aureus domains D, A, B and C that were analyzed (FIG. 11). The positionof the Q to K substitution in domain E (yellow), is located on the faceof Helix II that does not interact with the VH3 Fab from human IgM.

FIGS. 11A-11E show the amino acid sequence alignment of individual SpAIgBP domains A-E, respectively, from sequenced stains of S. aureus. Theposition of intra-SpA domain substitutions found in different S. aureusstains are indicated with an arrow. If the substitution is found in oneor more of the other SpA domains, it is indicated under the arrow. Forexample, the SpA A domain of S. aureus stain CC45_A9635 has and E to Dsubstitution at residue #8 (FIG. 11A). Residue D is found at thehomologous position of SpA domain D in all sequenced stains of S. aureus(amino acid position 11 in domain D).

FIG. 12 illustrates the domain structure of SpA and Sbi in Panel A.Domains I and II of Sbi have homology to SpA IgBP domains. Panel B showsthe amino acid sequence of domains E, D, A, B and C (Kim et al., 2010).Helix regions (see FIGS. 8 and 9) are labeled H1 (Helix I), 2 (Helix II)and 3 Helix III). The amino acid sequence of domains I and II of Sbi areshown. Similarity analysis of Sbi domains I and II are shown above theSbi sequences. Amino acids that are conserved between Sbi domains I andII and SpA IgBP domains are shown below the Sbi amino acid sequences.

FIG. 13 The IgFc binding domains of Sbi (I and II: FIG. 12) wereanalyzed. Amino acids within Helix I are highly conserved between Sbidomains I and II. Conservation is also found between Sbi domains I andII and SpA Fc binding domains within SpA helix I, and a number of aminoacids in SpA Helix II (* in FIG. 12B). Invariant residues (conserved inSpA domains and Sbi domains I and II) were mapped onto the model of Spadomain B (Helix I and II shown) binding to the Fc region of IgG.Important residues (yellow residues and arrows) that interact with Fcdomain are conserved between SpA domains and Sbi domains. In addition tothese invariant residues, a number of residues are found in Sbi domainsI and II that are present in some SpA domains (FIG. 12). Thus, the Fcbinding interface of Sbi and SpA has a high degree of conservation.

FIG. 14 shows the amino acid sequence of the individual Sbi IgBP domainsfrom sequenced stains of S. aureus. Panel A illustrates multiplesequence alignment of Sbi domain I. Panel B illustrates multiplesequence alignment of Sbi domain II.

FIG. 15 models the IgFc binding domains of Sbi (Domains I and II: FIG.14), using the structure of SpA domain B. Sbi was analyzed forinter-strain substitutions within domains I and II. One Sbi amino acidswithin Domain I of strain CC239_JKD6009 was found to differ. Thissubstitution (yellow residue and arrow in FIG. 15) is located in thepredicted Helix I of Sbi. This position is not conserved between Sbidomains I and II. The position of this substitution (amino acid N to Ssubstitution) was mapped onto the model of SpA domain B (Helix I and IIshown) binding to the Fc region of IgG. As shown, the residue (yellowresidue and arrow) is not predicted to form an interaction with the Fcdomain.

FIG. 16 is an SDS-PAGE and Western blot of anti-SpA parental antibodyMAB1 (upper panels) and anti-SpA-variant antibody MAB2 (Lower panels).

FIG. 17 is an SDS-PAGE and Western blot of anti-ClfA parental antibodyMAB3 (upper panels) and variant antibody MAB4 (Lower panels).

FIG. 18 is an SDS-PAGE and Western blot of anti-RSV variant antibodyMAB5.

FIG. 19 shows constructs used for DLS and immune-diffusion studies. Thefollowing S. aureus SpA and Sbi IgBPs proteins or their domains havebeen used to characterize S. aureus IgBP binding to variant and parentalheavy chain constant region sequences. Purified constructs have beenused for immunodiffusion and Dynamic Light Scattering experiments.

FIG. 20 shows an immuodiffusion analysis of antibodies MAB1, MAB2 andMAB5 with SpA and Sbi-E.

FIG. 21 shows an immuodiffusion analysis of antibodies MAB1, MAB2 withSpA domain D and Sbi-domains II/IV.

FIG. 22 shows an anti-RSV variant MAB5 analysis by DLS. The left panelshows the analysis of the control anti-RSV variant antibody alone(MAB5). The right panels show the size distribution in the presence ofeither Sbi-E (fragment of Sbi containing the two Ig-binding domains andtwo complement binding domains) or SpA1-4 (SpA IgBP domains 1-4).

FIG. 23 shows an anti-SpA parental MAB1 analysis by DLS. The left panelsshown the analysis of the parental anti-SpA antibody (MAB5) alone (upperpanel) or with Sbi III/IV (fragment of Sbi containing the two complementbinding domains (lower panel)). The right panels show the sizedistribution in the presence of either SpA 1-4 (SpA IgBP domains 1-4) orSbi-E (fragment of Sbi containing the two Ig-binding domains and twocomplement binding domains).

FIG. 24 illustrates a time dependent DLS peak shift with MAB1 and SpA-2.The left panel shown the analysis of the parental anti-SpA antibodyalone (MAB1). The left panels show the size distribution in the presenceof either SpA-2 (fragment of SpA containing domain D) after 1 min (upperpanel) or 10 mins incubation (lower panel).

FIG. 25 shows an anti-SpA variant MAB2 analysis by DLS. The left panelsshown the analysis of the variant anti-SpA antibody (MAB2) alone (upperpanel) or with Sbi-E (fragment of Sbi containing the two Ig-bindingdomains and two complement binding domains (lower panel)). The rightpanels show the size distribution in the presence of SpA 1-4 (SpA IgBPdomains 1-4). The lower left panel shows the overlap of the MAB2 controlplot (red) and the MAB2 plot in the presence of SpA 1-4 (green).

FIG. 26 shows an anti-SpA variant MAB2 analysis by DLS-Single SpAdomain. The upper panels shown the analysis of the variant anti-SpAantibody (MAB2) alone or with SpA-2 (SpA domain D).—lower panel.

FIG. 27 shows an analysis of binding of antibodies to S. aureus Newmanand a S. aureus SpA deletion stain in the absence and presence ofblocking human IgG1Fc. Antibodies tested include anti-SpA MAB1, anti-SpAvariant MAB2, anti-ClfA Parental MAB, anti-RSV variant MAB5 and anon-specific anti-KLH antibody. In the left panels, antibodies aretested in the absence of hIgG1 Fc. The upper panels represent binding toa S. aureus stain lacking expression of SpA (delta SpA=ΔSpA). The lowerpanels represent binding to S. aureus Newman stain. Blank contains noprimary antibody.

FIG. 28 shows tabulated results of ELISA binding of antibodies and theirvariants to a S. aureus stain lacking expression of SpA (Yellow panel:ΔspA) and to S. aureus Newman stain (Pink Panel: Newman) in the presence(Red bars) or absence (Blue bars) of human IgG1-Fc.

FIG. 29 shows a FACS analysis of antibody Binding to S. aureus: Bindingof the parental anti-SpA antibody (MAB1), an example anti-SpA variantantibody (MAB2) and a heavy chain constant region matched anti-RSVvariant control antibody (MAB6) were investigated by FACS, using S.aureus Newman strain (upper panels) or a ΔSpA strain (lower panels))grown to log phase (left panels) or from stationary phase cultures(right panels).

FIGS. 30A and 30B show antibody mediated C1q deposition: FACS analysiswas performed to test whether the parental anti-SpA antibody MAB1(center panel), its variant anti-SpA antibody MAB2 (right panel), or thecontrol anti-RSV variant antibody MAB5 (left panel) antibodies are ableto deposit C1q on wild type S. aureus Newman (FIG. 30A) or a S. aureusΔSpA stain (FIG. 30B). The upper panel of FIGS. 30(a) and (b) use ananti-hC1q detection antibody, while the lower panels use a negativecontrol detection antibody. Three concentrations of each antibody areshown for each Histogram: 3.33 μg/ml (red), 10 μg/ml (blue) and 30 μg/ml(green).

FIG. 31 shows a tabulation of antibody mediated C1q deposition data. Thedata from the FACS Histograms in FIG. 30 are tabulated. The Y axis showsstained bacterial population.

FIG. 32 shows a C3 complement deposition assay on Staph JE2 using FACS.C3 deposition on the surface of S. aureus stain JE2 was tested using theanti-SpA parental antibody (MAB1: Red plot) and an example anti-SpAvariant antibody (MAB2; Green Plot).

FIG. 33 illustrates neutrophil-mediated opsonophagocytic activity ofanti-SpA antibodies. The anti-SpA parental antibody (MAB1—Green plot)and an example variant anti-SpA antibody (MAB2—pink plot) were tested inphagocytosis assay using S. aureus Newman stain (left panel) and a ΔSpAstrain lacking SpA expression (right panel). Control anti-RSV variant(MAB5-Blue plot) and anti-KLH antibodies (Red Plot) are used as negativecontrols. Phagocytosis of FITC labeled S. aureus was analyzed by FACS.

FIG. 34 shows an opsonophagocytic assay of anti-SpA antibodies.Opsonophagocytic assay of MAB1 and MAB2 using S. aureus JE2 wasdetermined in a second Opsinophagocytic assay. % Bacterial survival (Yaxis) was measured for S. aureus JE2 following treatment with MAB1 (darkbars) and MAB 2 (light bars) using 5 ug/ml and 25 ug/ml of testantibody. A control (no antibody) is shown on the left side (grey bar)

FIG. 35 shows a neutrophil-mediated opsonophagocytic bactericidal assay.Opsonophagocytic killing of S. aureus JE2 using pooled human serum.Parental anti-SpA MAB1 (Dark Bars) and an example variant anti-SpA MAB2(light bars) were tested for their effect on the opsonophagocytickilling of S. aureus JE2 using 5 μg/ml (left bar of pair) and 25 μg/ml(right bar of pair) of test antibody.

FIG. 36 shows the amino acid sequence of the light chain (SEQ ID NO:58)and heavy chain SEQ ID NO:59) of a CS-D7 parental monoclonal antibody.

FIG. 37 shows the amino acid sequence of three light chains (SEQ IDNOs:60-62) and three heavy chains (SEQ ID NOs:63-65) of an anti-LTAparental monoclonal antibody.

DETAILED DESCRIPTION

Antibodies and variant antibodies that may be used to reduce, treat oreliminate pathogenic microbes and/or associated virulence factors areprovided herein. The variant antibodies described herein have enhancedantimicrobial activity relative to their parent antibody. In someembodiments, the enhanced antimicrobial activity is due to at least oneamino acid substitution—as compared to the parent antibody—that givesrise to a variant heavy chain constant region, a variant variableregion, or both. As described below, such variant constant and variableregions may result in attenuated non-immune binding to one or morevirulence factors produced by a targeted pathogenic microbe. Virulencefactor binding that may be affected by the variant heavy chain constantand heavy chain variable regions described herein include virulencefactors that involve non-immune binding to an antibody (e.g.,immunoglobulin binding proteins (IgBPs), Fc binding proteins (FcBPs) andsuperantigens which interact with the Fab domain). Variant antibodies ofthe invention are directed against microbial antigens, includingvirulence factors and IgBPs. Antibodies and variant antibodies describedherein can be affinity matured, resulting in enhanced antigen specificimmune binding.

Microbial Targets

The antibodies and variant antibodies described herein may be designedto target the effector function of any pathogenic microbe including, butnot limited to, bacteria (e.g., bacteria from the following genare:Bordetella, Borrelia, Brucilla, Campylobacter, Chlamydia, Chlamydophila,Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella,Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio,Yersinia), viruses (e.g. Adenovirus, Coxsackievirus, Epstein-Barr virus,Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Herpes simplexvirus, type 1, Herpes simplex virus, type 2, cytomegalovirus, Humanherpesvirus, type 8, HIV, Influenza virus, measles virus, Mumps virus,Human papillomavirus, Parainfluenza virus, Poliovirus, Rabies virus,Respiratory syncytial virus, Rubella virus, Varicella-zoster virus), andparasites (e.g., Acanthamoeba, Anisakis, Balantidium coli, Entamoebahistolytica, Giardia lamblia, Leishmania, Plasmodium falciparum,Schistosoma, Toxoplasma gondii, Trypanosoma).

In certain embodiments, the antibodies and variant antibodies may bedesigned to target the pathogenic gram positive bacteria, such asStaphylococci aureus (S. aureus) and group A Streptococcus (GAS). S.aureus and GAS are prominent Gram-positive human pathogens responsiblefor a wide spectrum of superficial and invasive disease conditions(Nizet, 2007). S. aureus accounts for >10 million skin and soft tissueinfections annually in the United States alone1 and is the singleleading cause of hospital acquired infections. Each year worldwide, GASis responsible for more than 700 million cases of pharyngitis or skininfection and more than 650,000 invasive infections. Both pathogens canproduce infections in essentially every human organ or tissue, includingsevere life-threatening conditions such as necrotizing fascitis,endocarditis, sepsis, and toxic shock syndrome. The propensity of S.aureus and GAS to produce systemic infections, often in otherwisehealthy children and adults, defines a capacity of each pathogen toresist host immune clearance mechanisms that normally function toprevent microbial dissemination beyond epithelial surfaces. S. aureusand GAS systemic disease reflects diverse abilities of these pathogensto resist clearance by the multifaceted defenses of the human immunesystem. The mechanisms by which S. aureus and GAS avoid the bactericidalactivities of cationic antimicrobial peptides, delay phagocyterecruitment, escape neutrophil extracellular traps, inhibit complementand antibody opsonization functions, impair phagocytotic uptake, resistoxidative burst killing, and promote phagocyte lysis or apoptosis havebeen reviewed by Nizet (Nizet, 2007).

S. aureus causes a variety of suppurative (pus-forming) infections andtoxinoses in humans. It causes superficial skin lesions such as boils,styles and furuncules; more serious infections such as pneumonia,mastitis, phlebitis, meningitis, and urinary tract infections; anddeep-seated infections, such as osteomyelitis and endocarditis. S.aureus is a major cause of hospital acquired (nosocomial) infection ofsurgical wounds and infections associated with in dwelling medicaldevices. S. aureus causes food poisoning by releasing enterotoxins intofood, and toxic shock syndrome by release of superantigens into theblood stream. Although methicillin-resistant S. aureus (MRSA) have beenentrenched in hospital settings for several decades, MRSA strains haverecently emerged outside the hospital becoming known as communityassociated-MRSA (CA-MRSA) or superbug strains of the organism, which nowaccount for the majority of staphylococcal infections seen in the ER orclinic. S. aureus permanently colonizes the moist squamous epithelium ofthe anterior nares of 20% of the population, and is transientlyassociated with another 60%. Occasionally, the organism can causesuperficial skin infections such as abscesses and impetigo, or seriousinvasive infections such as septic arthritis, osteomyelitis andendocarditis. Colonization is a known risk factor for invasive diseaseboth in the hospital and the community. Hospital patients who have beencatheterized or who have undergone surgery are at increased risk ofinfection. Treatment of infections with antibiotics has becomeincreasingly difficult owing to the widespread occurrence of strainsthat are resistant to multiple antibiotics, known as meticillin(formerly methicillin)-resistant S. aureus (MRSA). Furthermore, theisolation of MRSA strains that have also become resistant to vancomycin,the last drug to which the organism had been uniformly sensitive, raisesthe spectre of a return to the pre-antibiotic era.

The pathogenicity of S. aureus is a complex process involving a diversearray of extracellular and cell wall components that are alsocoordinately expressed during different stages of infection (i.e.colonization, avoidance of host defense, growth and cell division,bacterial spread). The coordinated expression of diverse virulencefactors in response to environmental cues during infections (e.g.expression of adhesins early during colonization vs. production oftoxins late in infection to facilitate tissue spread) suggests theexistence of global regulators in which a single regulatory determinantcontrols the expression of many unlinked target genes (FEMS Immunol MedMicrobiol. 2004 Jan. 15; 40(1):1-9). Bacteria use quorum sensing tosynchronize release of these molecules. Individual bacteria secretesmall molecules termed “auto-inducers” (AI), including N-acylhomo-serine lactones (gram-negative bacteria) and oligopeptides(gram-positive bacteria), at a constant, low level as a means ofdetecting the local concentration of bacteria.

An individual who has suffered from a S. aureus infection is usually notprotected from a subsequent infection. This is because the host isprevented from mounting a strong antibody response, and immunologicalmemory is compromised by the immunosuppressive activities of Vβ bindingsuperantigens (Enterotoxin B, TSST-1, SAC1-3) and by nonspecificpolyclonal B cell receptor activation resulting from binding of SpA tothe B cell receptor Ig heavy-chain gene products of the VH3 family. Inone such embodiment, the antibodies may be designed to targetStaphylococci aureus (S. aureus).

Virulence Factors and their Effect on the Immune Response

The antimicrobial effector functions of antibodies are the target of anumber of microbial immune evasion strategies, which have evolved toevade the immune response to the pathogen. These molecules, which areresponsible for immune evasion, belong to the family of microbialvirulence factors.

An immune response to a foreign antigen includes the production ofantibodies by B-cells of the immune system against the foreign antigensthat are detected within the body. Pathogenic microbes are one source offoreign antigens, which can stimulate the production of antibodies. Asuccessful humoral immune response against such a pathogen results inthe production of high affinity antibodies against the microbe, whichare able to contribute to the elimination of the infection due to theeffector function of the antibody. However, as described in detailbelow, a variety of pathogenic microbes (e.g., Staphylococci andStreptococci) produce a variety of virulence factors, which, among otherfunctions, are able to attenuate the immune response against themicrobe.

Virulence factors refer to microbial proteins or molecules (i.e., geneproducts) that enable a pathogen to establish itself on or within a hostof a particular species and enhance its potential to cause disease. Mostbacterial virulence genes involved in pathogenesis encode proteins thatare either displayed on the bacterial surface (e.g. cell surfaceproteins) or are released into the surroundings (e.g. toxins andenzymes) and confers resistance to antimicrobials, which may strengthenits virulence and confer resistance to all families of antibiotics.These enable the organism to evade host defenses, to adhere to cells andthe tissue matrix, to spread within the host and to degrade cells andtissues, for both nutrition and protection. These accessory genes arecollectively known as the virulon (Novick 2003).

In one embodiment, a variant antibody has attenuated binding to avirulence factor when it binds the virulence factor in a non-immunefashion, i.e., the antibody binds the one or more virulence factor via aportion of the antibody that is not involved in antigen-dependentbinding to the variable region of the antibody (e.g., Fc region bindingor superantigen type Fab binding). In another embodiment, an antibody orvariant antibody has enhanced antigen dependent immune-binding to avirulence factor when it binds the virulence factor in an immunefashion, i.e., the antibody or variant antibody binds a virulence factorin an antigen-dependent manner, via binding to the variable region ofthe antibody. Such antibodies and their variants can undergo affinitymaturation when their antigen-dependent binding is enhanced by one ormore amino acid substitution within the variable domain. These alteredbinding properties of the variant antibodies are not mutually exclusive.In other words, a variant antibody may have one or more amino acidsubstitutions (described below) that result in both attenuatednon-immune binding to one or more IgBP virulence factors, and enhancedimmune-binding to one or more virulence factors or microbial antigens.

Microbial proteins that contribute to the virulence of microbes such asS. aureus can be divided into at least five major families (describedbelow): immune response inhibitors, superantigens, adhesion proteins,toxins, and enzymes. These virulence factor families are describedbelow.

Immune Response Inhibitors.

Many bacteria produce virulence factors that inhibit the host's immunesystem defenses. For example, a common bacterial strategy is to produceIgBPs that bind to the Fc region of host immunoglobulin. Such IgBPs bindto the Fc region via non-immune binding. The best studies examples ofFcBPs are SpA and Protein G (Goward et al., 1993; Bjorck et al., 1984,Reis et al., 1984), which are described in detail below. Additionally,the polysaccharide capsule coating the microbe can prevent effectiveelimination by host immune system effector cells.

Microbial IgBPs.

The non-immune binding of antibodies by bacterial or viralimmunoglobulin IgBPs and FcBPs is a common strategy employed bypathogens to subvert the host immune response. The capacity ofmicroorganisms to bind IgG or IgA via the Fc region has emerged as awidespread biological phenomenon. Immunoglobulin FcBPs are expressed onthe cell surface of many Gram-positive pathogenic bacteria and are ableto bind to Igs in a non-immune manner. In many cases, the Fc bindingfunction has been incorporated into a larger multifunctional protein,which can have a number of virulence functions. The role of theseproteins is almost certainly to help the bacteria adhere to hostsurfaces and to evade the immune response by allowing host proteins tocover the bacterial cell surface. Two of the first such proteins to berecognized were SpA (also referred to as Protein A), a cell-wallcomponent of S. aureus, and Protein G, a protein associated with thecell walls of certain Streptococci. The Fc Binding Protein, SpA of S.aureus, serves to block Fc-receptor mediated effector functions such asopsonophagocytosis and contributes to virulence (Palmqvist et al.,2002). SpA binds the Fc region of IgG (Uhlen et al., 1984) and preventsboth opsonophagocytosis and complement fixation (Gemmell et al., 1991).

Such FcBP proteins are also found among bacteria including streptococciof groups A, C, and G (Kronvall, 1973; Schroder et al., 1986), andStaphylococci (Forsgren & Sjouist, J Immunol. 1966). Furthermore, IgGFcBPs have been demonstrated on cells infected with Herpes simplex virus(Watkins, 1964; Chapman et al., 1999) of both serotypes I and 2 (Para,1982), Varicellazoster (Ogata & Shigeta, 1974), Epstein-Barr virus (Yeeet al., 1982) and Cytomegalovirus (Furatawa et al., 1975; Keller et al.,1976; Sprague et al., 2008; Lilley et al., 2001), Hepatitis C virus(Maillard et al., 2004), as well as on cells infected with schistosomes(Torpier, 1979).

Examples of such Fc Binding proteins (reviewed in Sidorin and Solov'eva,2011) include, but are not limited to, Protein G from group C and groupG streptococci (Derrick, 1992; Bjorck, 1984; Reis et al., 1984); βprotein and M or M like family FcBPs such as Protein H, Arp4, Arp 60,Mrp4, Sir22, Enn4, FgBP (Kazeeval & Shevelev, 2009; Pleass et al., 2001;O'Toole et al., 1992; Heden et al., 1991; Jerlstro et al., 1991; Lewiset al., 2008; Meehan et al., 2001); SibA from GAS (Fagan et al., 1991);SpA from S. aureus (Boyle 1990 in Bacterial Immunoglobulin-BindingProteins, ed. Boyle, M. P. D. (Academic, San Diego), Vol. 1, pp. 17-28,Gouda et al., Biochemistry. 1992 31:9665-7; Goward et al., 1993); SSLfamily members including SSL 7 and SSL10 from S. aureus (Ramsland etal., 2007; Kazeeval & Shevelev 2009; Ramsland et al., 2007); Sbi from S.aureus (Burman et al., 2008, Itoh et al., 2010); IsaB from S. aureus(Clark et al., 1999); PsaA from Y. Pestis; Eib-proteins from E. Coli(Sidorin. and Solov'eva, 2011).

It has been demonstrated that there is a striking overlap of the Ig Fcregions recognized by distinct bacterial IgBPs proteins, despite thefact that they derive from very different microbes (e.g. streptococcaland staphylococcal strains) and these microbes are pathogenic indifferent mammalian species. Even more remarkably, the fact thatunrelated IgG binding proteins (e.g. SpA, Protein G) bind to similarsites in the IgG interdomain region parallels the situation for theunrelated bacterial IgA binding proteins (e.g. Sir22 from S. pyogenes,β-protein from group B streptococcus, and SSL7 from S. aureus), whichall bind to the Fc domain interface in human IgA (Pleass et al., 2001;Wines et al., 2006; Ramsland et al., 2007). Although a differentimmunoglobulin class is involved, FcBP proteins produced by verydifferent bacterial pathogens target the equivalent Fc region. Thus, itseems that convergent evolution may have favored the appearance ofbacterial proteins that bind to the CH2/CH3 interface in IgG and IgA.This interdomain region in IgG, has been recognized as one of only alimited number of regions on the Ig surface that is particularly suitedto protein-protein interactions (Burton, 1985; DeLano et al., 2000). Forexample, purified IgG Fc binding protein (FcBP) from the M15 strain ofgroup A streptococci binds to the same site in the interface between theCH2 and CH3 domains as SpA, Protein G (SPA) (Nardella et al., 1985;Nardella et al., 1987) and IgG rheumatoid factors. His 435 and Tyr 436on the IgG heavy chain, and possibly one or both of His 433 and 310,were demonstrated to be involved in the binding. The importance of His435 in binding of many FcBPs to IgG originated with the findings on thespecificity of IgG isotypes and allotypes for SpA. It was found thathuman IgG3 allotypes with Arg at position 435 lack the ability to bindSpA (Recht et al., 1982; van Loghem et al., 1982). IgG3 allotypes andIgG isotypes capable of binding SpA possess a His residue at position435 within the interaction site for SpA. However, allotypes, which carryan Arg at this important CH3 domain residue, are unable to bind SpA(Recht et al., 1982; van Loghem et al., 1982).

Lack of Fc binding of antibodies to SpA has been used to develop anumber of applications, including antibody based diagnostic tests for S.aureus Infection (Larsson & Sjoquist, 1989). In such cases, chicken ormouse immunoglobulin of non-binding isotypes can be used for theselective testing for S. aureus SpA by immune-assays.

The evolutionary reasons why such sites of relative vulnerability havebeen retained on the surface of Ig Fc regions probably relate to theirrole as interaction sites for important host receptors. In IgG, forexample, the Fc interdomain region forms the interaction site for FcRn,the so-called neonatal Fc receptor that mediates a number of processesfundamental to IgG function, including regulation of IgG turnover andtransepithelial transfer of IgG. It has been shown that the same residueat position 435 is important for FcRn binding. Its mutation (H435A)results in loss of binding of the antibody to both human and mouse FcRn.

Superantigens.

Superantigens (SAgs) are a class of antigens, which cause non-specificactivation of T-cells or B-cells, resulting in polyclonal T or B cellactivation. SAgs can be produced by pathogenic microbes (includingviruses, mycoplasma and bacteria) (Llewely, 2002) as a defense mechanismagainst the immune system, and bind to antibodies via non-immunebinding.

Superantigens are microbial or viral toxins that comprise a class ofdisease-associated, immunostimulatory molecules and act as Vβ-restrictedextremely potent polyclonal T cell mitogens. They bind majorhistocompatibility complex (MHC) class-II molecules without any priorprocessing and stimulate large number of T cells (up to 20% of all Tcells) on the basis of epitope specified by this receptor (Papageorgiou& Acharya, 2000; Acharya et al., 1994; Haynes & Fauci 2005). Theseproperties are attributable to their unique ability to cross-link MHCclass II and the T cell receptor (TCR), forming a trimolecular complex.The large number of activated T-cells generates a massive immuneresponse, which is not specific to any particular epitope on the SAgthus undermining one of the fundamental strengths of the adaptive immunesystem, that is, its ability to target antigens with high specificity.More importantly, the large numbers of activated T-cells secrete largeamounts of cytokines, which can cause severe and life-threateningsymptoms, including shock and multiple organ failure.

In contrast, B cell-directed superantigens target the B cellcompartment. By definition, these agents (1) stimulate a high frequencyof B cells, (2) target B cells that have restricted usage of VH or VLfamily genes, and (3) bind to immunoglobulins outside the sites thatbind conventional antigens. A B-cell superantigen that has receivedconsiderable attention is staphylococcal SpA (Silverman et al., 2000;Graille et al., 2000). This agent has the ability to bind to the Fcfragment of IgG. This binding has been localized to a region containsα-helical 1 and 2 (helix I and II) structures on each of four or fivehomologous regions that comprise the extracellular domain of SpA (FIG.2). However, it is now clear that SpA repeat IgG binding domains containa second site, located in a region containing helix II and helix III,(FIG. 2) that binds to determinants on the Fab regions of certainimmunoglobulins independently of their heavy-chain isotype (Graille etal., 2000). In humans, this so-called alternative site appears to bindonly to immunoglobulins that utilize heavy-chain genes of the VH3subfamily. The x-ray structure of this interaction has been solved,explaining the basis for this interaction (FIG. 3). In the mouse thistype of binding is restricted to immunoglobulins using heavy chainsbelonging to the S107 and J606 VH families.

A number of microbial immunoglobulin binding proteins (IgBP) can bind toregions of immunoglobulin outside the Fc region. Examples of suchproteins include SpA, which is also able to bind to the Fab region ofMost VH3 sequences. This binding uses a separate binding site to thatused for Fc binding. The ability to bind to Fab sequences enables SpA toact as a B cell superantigen. The L protein from the surface ofbacterial species Peptostreptococcus magnus was found to bind Ig throughL chain interaction, from which the name was suggested (Bjorck, 1988).Unlike SpA and Protein G, which bind to the Fc region of immunoglobulins(antibodies), Protein L binds antibodies through light chaininteractions. Since no part of the heavy chain is involved in thebinding interaction, Protein L binds a wider range of antibody classesthan SpA or G. Protein L binds to representatives of all antibodyclasses, including IgG, IgM, IgA, IgE and IgD. Single chain variablefragments (ScFv) and Fab fragments also bind to Protein L.

Despite this wide binding range, Protein L is not a universalantibody-binding protein. Protein L binding is restricted to thoseantibodies that contain kappa light chains. In humans and mice, mostantibody molecules contain kappa (κ) light chains and the remainder havelambda (ι) light chains. Protein L is only effective in binding certainsubtypes of kappa light chains. For example, it binds human VκI, VκIIIand VκIV subtypes but does not bind the VκII subtype. Binding of mouseimmunoglobulins is restricted to those having VκI light chains (Nilsonet al., 1993).

Adhesion Proteins.

Many bacteria must first bind to host cell surfaces. Many bacterial andhost molecules that are involved in the adhesion of bacteria to hostcells have been identified. Often, the host cell receptors for bacteriaare essential proteins for other functions. Members of the MSCRAMM(microbial surface component recognizing adhesive matrix molecule)family of adhesion proteins bind ECM ligands such as collagen,fibronectin, and fibrinogen,

Toxins.

Many virulence factors are proteins made by bacteria that poison hostcells and cause tissue damage. For example, there are many foodpoisoning toxins produced by bacteria that can contaminate human foods.Some of these can remain in “spoiled” food even after cooking and causeillness when the contaminated food is consumed

Enzymes.

A number of virulence factors encode proteases or microbial activatorsof host protease, which are able to interfere with antibody andcomplement mediated microbial killing. For example, S. aureus canexpress a number of proteases or zymogens, and additional virulencefactors, which encode enzymes such as lipases, deoxyribonucleases(DNase) and a fatty acid modifying enzymes.

Microbial Antigenic Surface Proteins

Surface proteins of S. aureus are linked to the cell wall by sortase, anenzyme that cleaves polypeptides at a conserved LPXTG motif. SpA, asurface protein of S. aureus synthesized as a precursor bearing anN-terminal signal peptide, which is cleaved during secretion, and aC-terminal sorting signal with an LPXTG motif. After signalpeptide-mediated initiation of the precursor into the secretory pathway,the sorting signal directs SpA to the cell wall envelope. Thepolypeptide is then cleaved between the threonine and the glycine of theLPXTG motif. The liberated carboxyl group of threonine forms an amidebond with the amino group of the pentaglycine crossbridge, therebytethering the C terminus of SpA to the bacterial peptidoglycan. Thegenome of S. aureus encodes at least 10 different surface proteinsbearing C-terminal sorting signals with an LPXTG motif. Many of thesepolypeptides are known to interact with various human tissues, serumproteins, or polypeptides of the extracellular matrix. For example, SpAbinds to the Fc portion of immunoglobulins, a mechanism that is thoughtto prevent opsonophagocytosis of staphylococci after their entry intothe human host. Binding of the clumping factors, ClfA and ClfB, tofibrinogen promotes bacterial adhesion to vascular and endocardiclesions. The FnbA and FnbB surface proteins bind to fibronectin. Thisinteraction allows staphylococci to adhere to various tissues and,similar to fibronectin-binding proteins of Streptococcus pyrogenes,presumably provides for the invasion and apoptotic death of infectedepithelial cells.

According to the embodiments described herein, anti-microbial monoclonalantibodies and variant monoclonal antibodies that have variable domainsthat recognize one or more microbial cell surface or secreted antigensare provided.

In some embodiments, IgG antibodies, such as a human IgG antibody, ahumanized or a chimeric IgG class antibody or their variants areprovided. In such embodiments, the antigen recognition region of theantibody is directed against one or more microbial cell surface orsecreted antigens (i.e., antigen specific immune binding). In someembodiments, the one or more microbial cell surface or secreted antigensinclude ClfA, SpA and Sbi.

In other embodiments, IgG antibodies, such as a non-human IgG antibody,or their variants are claimed for use in veterinary medicine. In suchembodiments, the antigen recognition region of the antibody is directedagainst one or more microbial cell surface or secreted antigen.

Virulence Factors of a Target Microbe, S. aureus

In some embodiments, the antibodies and variant antibodies describedherein may be designed to target one or more virulence factors producedby the target microbe, which according to some aspects, is S. aureus. S.aureus produces an array of virulence factors (Foster, 2005), examplesof which include (1) cell surface proteins that promote colonization ofhost tissues (e.g. SpA (Protein A), Clumping Factor A (ClfA); (2)invasins that promote bacterial spread in tissues (e.g. leukocidin,hyaluronidase); (3) cell surface factors that inhibit phagocyticengulfment and complement mediated killing (SpA, Sbi, CapsularPolysaccharide Serotypes 5 and 8 (Cps 5 and 8); (4) biochemicalproperties that enhance their survival in phagocytes (proteases, andprotease activators: among the array of secreted staphylococcal factors,a number of proteases, including the serine proteases V8 (SspA/V8) andSpIA-SpIF, the cysteine proteases ScpA (staphopain A) and SspB(staphopain B), the metalloprotease aureolysin and, staphylokinase whichcan activate host zymogens; (5) immunoglobulin binding proteins (SpA,Sbi, SSL10, SSL7); (6) membrane-damaging toxins that lyse eukaryoticcell membranes (e.g. γ-hemolysins, leukotoxin E-D, Panton-Valentinleukocidin; (7) exotoxins that damage host tissues or otherwise provokesymptoms of disease (e.g. Enterotoxins A, B, C, D, G, H); (8)superantigens which compromise the T cell or B cell response (e.g. SpA,Enterotoxin B, TSST-1, SAC1-3); and (9) inherent and acquired resistanceto antimicrobial agents. Several of the virulence factors that may beaffected by the variant antibodies described herein are described below.

SpA.

SpA (Protein A), which exists in both secreted and membrane-associatedforms, possesses two distinct Ig-binding activities: each domain canbind Fcγ (the constant region of IgG involved in effector functions, asdescribed above) and Fab (the Ig fragment responsible for antigenrecognition) (Boyle, 1990). SpA is a 42-kDa protein covalently anchoredin the staphylococcal cell wall through its carboxyl terminal end. Theprotein is comprised of five repeated domains (E, D, A, B, C) of ˜58residues linked to the cell surface by region Xr, which contains avariable number of short 8-residue repeats (FIG. 2). Each SpA domain canbind with high affinity to the Fc region of immunoglobulin G and to theFab region of immunoglobulin of the VH3 subclass (Jansson et al., 1998,Moks et al., 1986; Roben et al., 1995; Sasso et al., 1989). Theinteraction with IgG Fc hinders phagocytosis because bacteria becomecoated with IgG in an inappropriate conformation not recognized by theFc receptor on neutrophils. Moreover, SpA-bound IgG cannot stimulatecomplement fixation by the classical pathway. An additional consequenceof the ability of SpA to bind to B lymphocytes displaying IgM bearingVH3 heavy chains is the induction of proliferation resulting indepletion of a significant part of the B cell repertoire (Goodyear etal., 2004; Viau et al., 2005).

Both the SpA-Fc and SpA-Fab interactions have been analyzed at themolecular level with co-crystallized complexes (Deisenhofer 1981; Goudaet al., 1998; Graille et al., 2000). The SpA domains adopt three-helixbundles. One face includes residues from helices I and II binds IgG Fc,whereas residues from helices II and III on the other face bind VH3 Ig(Graille et al., 2000). The residues from helix II that bind Fc aredifferent from those that bind Fab, with the exception of a singleglutamine (Gln-32 in SpA domain D) (Deisenhofer 1981; Graille et al.,2000). SpA also binding strongly to a number of other proteins includingvon Willebrand factor (vWF) (O'Seaghdha et al., 2006), the TNF receptorI (TNFRI) (Gomez et al., 2006), the Epidermal growth factor receptor(EGFR) (Gomez et al., 2007) and also binds to an undefined target onosteoblasts (Claro et al., 2011).

The SpA Fcγ binding site has been localized to the elbow region at theCH2 and CH3 interface of most IgG subclasses, and this binding propertyhas been extensively used for the labeling and purification ofantibodies (Deisenhofer, 1981; Tashiro & Montelione, 1995). The X-raystructure of the SpA IgG-binding domains in complex with the Fc regionof IgG have been solved and residues from helix I and II that areinvolved in the interaction have been identified and verified bysite-directed mutagenesis, and by the existence of allotypes of IgG3(with an Arg435 residue) that do not bind SpA. The consequence of theinteraction between SpA and IgG-Fc is to coat the surface of the cellwith IgG molecules that are in the incorrect orientation to berecognized by Fc receptors on effector cells. This could explain theanti-phagocytic effect of SpA and its role in the pathogenesis of S.aureus infections. Protein-A-deficient mutants of S. aureus arephagocytosed more efficiently by neutrophils in vitro and show decreasedvirulence in several animal infection models (Gemmell et al., 1997;Palmqvist et al., 2002).

SpA (Protein A) also acts as a B-cell superantigen through interactionswith the heavy-chain variable part of Fab fragments, and sequestersimmunoglobulins by forming insoluble immune complexes with human IgG. Ithas been shown that the formation of insoluble immune complexes ismediated by the binding of (VH3+) Fab fragments in addition to Fc.B-cell superantigens, unlike conventional antigens, bind to the Fabregions of immunoglobulin (Ig) molecules outside theircomplementarity-determining regions (CDRs) reviewed in references(Levinson et al., 1995; Silverman, 1997). These unconventional antigenscan react with a substantial amount of a host's peripheral B-cellrepertoire and serum Igs by virtue of their ability to interact withmany members of an entire variable region heavy (VH) or variable regionlight (VL) gene family (Levinson & Kozlowski, 1996).

S. aureus SpA (Protein A) is one of the most studied B-cell SAg.Although it had long been known that this microbial product binds to theFc region of IgG, it became clear that SpA also binds, via analternative site, to determinants outside the CDRs in the Fab region ofIgs. SpA reacts with the Fabs of most VH3 Igs, which are expressed on 30to 60% of human peripheral B cells. The crystal structure of an S.aureus SpA domain complexed with a Fab fragment of human IgM has beensolved, showing the molecular basis for B-Cell receptor recognition andsuperantigen activity. The interactions of SpA with the Fab region ofmembrane-anchored Igs can stimulate a large fraction of B cells,contributing to lymphocyte clonal selection. The crystal structure ofthe complex between domain D of SpA and the Fab fragment of a human IgMantibody to 2.7-Å resolution has been solved (Graille et al., 2000). Inthe complex, helices II and III of domain D interact with the variableregion of the Fab heavy chain (V_(H)) through framework residues,without the involvement of the hypervariable regions implicated inantigen recognition. The contact residues are highly conserved in humanV_(H)3 antibodies but not in other families. The contact residues fromdomain D also are conserved among all SpA Ig-binding domains, suggestingthat each could bind in a similar manner. Correlation with antibodysequence usage indicates that the Fab binding specificity is restrictedto products of the human variable region of the Fab heavy chain V_(H)3family that represent nearly half of inherited V_(H) genes (Sasso etal., 1989; Sasso et al., 1991; Sasano et al., 1993; Hillson et al.,1993) and their homologues in other mammalian species (Seppala et al.,1990; Cary et al., 1999). Presumably through interactions with surfacemembrane-associated V_(H)3-encoded B-cell antigen receptors (Romagnaniet al., 1982), in vitro stimulation with SpA can contribute to selectionof these B cells and promote their production of antibodies that mayinclude rheumatoid factor autoantibodies (Kristiansen et al., 1994);Kozlowski et al., 1995). In vivo exposure to recombinant SpA can resultin supraclonal suppression and deletion of B-lymphocytes that aresusceptible based on their V_(H) usage (Silverman et al., 1998; Cary etal., 2000).

Although the mechanism(s) are not defined, experimental models indicatethat SpA enhances staphylococcal virulence (Foster et al., 1988; Patelet al., 1987). Many features of the interactions of SpA with host Blymphocytes are akin to those of superantigens for T lymphocytes thatcause a variety of inflammatory diseases including toxic shock syndrome,food poisoning, and exfoliative syndromes (Kotzin et al., 1993; Bohachet al., 1990; Papageorgiou et al., 1998) and T-cell superantigens alsohave been postulated to contribute to the pathogenesis of autoimmunedisease (Li et al., 1999). These superantigens target T-cell receptors(TcRs) from particular variable β chain (V_(β)) families and induceglobal changes in T lymphocyte repertoires (Kotzin et al., 1993). Thesite responsible for Fab binding is structurally separate from thedomain surface that mediates Fcγ binding. As first demonstrated in acrystallographic complex and recently reinvestigated in NMR studies theinteraction of Fcγ with domain B primarily involves residues in helix Iwith lesser involvement of helix II (Graille et al., 2000). With theexception of the Gln-32, a minor contact in both complexes, none of theresidues that mediate the Fcγ interaction are involved in Fab binding.The area buried in the Fcγ-domain B interface is 1,320 Å², which iscomparable to the 1,220 Å² buried in the current complex with Fab.However, the nature of these buried SpA residues differs significantly,as the Fab binding is dominated by polar contacts whereas the Fcγinteraction is predominantly hydrophobic. To examine the spatialrelationship between these different Ig-binding sites, the SpA domainsin these complexes were superposed (Graille et al., 2000) to construct amodel of a complex between a Fab, a SpA domain, and an Fcγ molecule. Faband Fcγ form a sandwich about opposite faces of the helix II withoutevidence of steric hindrance of either interaction. These findingsillustrate how, despite its small size (i.e., 56-61 aa), SpA domains cansimultaneously display both activities, explaining experimental evidencethat the interactions of Fcγ and Fab with an individual domain arenoncompetitive (Starovasnik et al., 1999).

SpA has also been found to activates tumor necrosis factor receptor 1(TNFR1) (Gomez et al., 2004) Staphylococci frequently cause pneumonia,and these clinical isolates often have increased expression of SpA,suggesting that this protein may have a role in virulence. It has beenfound that TNFR1, a receptor for tumor-necrosis factor-α (TNF-α) that iswidely distributed on the airway epithelium, is a receptor for SpA(Gomez et al., 2004).

SpA can also act directly as an immune effector itself through itsability to bind and activate tumor necrosis factor α (TNF-α) receptor 1(TNFR1) (Gomez et al., 2004, 2006). This interaction is particularlyimportant at sites of infection where TNF-α signaling is important, asin the lung. SpA-TNFR1 interaction is essential for the pathogenesis ofpneumonia as TNFR1 null mice are not susceptible to S. aureus pneumoniaand SpA-defective mutants of S. aureus do not cause infection inwild-type animals. SpA activates proinflammatory signaling throughbinding to TNFR1 and activation of TRAF2, the p38/c-Jun NH2-terminalkinase MAPKs, and NF-κB (Gomez et al., 2004). TNFR1 ectodomain sheddingis induced by SpA (Gomez et al., 2004), presumably by activating theTNF-converting enzyme (TACE or ADAM17) through a signaling pathway notyet elucidated. As there is no apparent homology between the trimericTNFR1 and IgG, both of which function as receptors for SpA, we wereinterested in defining the molecular basis for the SpA-TNFR1interaction.

Each SpA binding domain includes a triple helical bundle (Deisenhofer1981). By analyzing a series of amino acid substitutions in the SpA Ddomain, Gomez et al (2006) showed that the residues important in theinteraction between SpA D and the Fc region of IgG are also involved inbinding to and activating TNFR1. SpA residues that are on the oppositeface of the protein that are involved in IgM Fab binding are notinvolved in the interaction with TNFR1 (Gomez et al., 2006). The IgG Fcregion binds to residues exposed on the face formed by helices I and II.TNFR-1 also binds to this face but there are some differences in theresidues of SpA that are involved. In particular, leucine 17 is crucialfor binding to IgG but not for TNFR-1 binding.

SpA is known to bind human von Willebrand factor (VWF), a protein thatis essential for haemostasis, with an affinity of 15 nM as measured bysurface plasmon resonance using full length recombinant SpA and VWF thathad been purified from plasma. This interaction was shown to occur inthe presence of physiological IgG concentrations. Heritable defects inVWF result in von Willebrand's disease, a common bleeding disorder,symptoms of which can mirror severe hemophilia. The main function of VWFis to capture platelets by binding to the platelet receptor GPIb-a andimmobilize them at the site of damage to a blood vessel and to stimulatethe formation of a blood clot. The VWF protein consists of four types ofrepeat domain A, B, C and D. Domains are arranged in the sequenceD′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK in the mature protein (for review,see Sadler J. E, 1998). The crystal structure of the recombinant A1domain in complex with platelet glycoprotein Gibe has been solved(Emsley et al., 1998; Huizinga et al., 2002; Uff et al., 2002). Bindingof circulating VWF to the ligands such as collagen in exposedsubendothelial matrix of damaged blood vessels under high shear-stressstimulates a conformational change which promotes immobilized VWFbinding to GpIba on platelets (Siedlecki et al., 1996; Novak et al.,2002; Hulstein et al., 2005). Circulating platelets are captured andactivated, stimulating the formation of a thrombus (Kroll et al., 1996;Xiong et al., 2003). The ability of S. aureus to bind VWF couldcontribute to the adherence of the bacterium to platelets or to damagedblood vessels. By studying a Spa-deficient mutant of S. aureus it wasshown that the Spa-VWF interaction is necessary for efficientrecruitment of S. aureus by platelets under high shear stress in wholeblood (Pawar et al., 2004). Also fluid-shear adhesion experimentssuggested that VWF binding to Spa can promote adherence of circulatingS. aureus cells to immobilized collagen (Mascara et al., 2003). In arecent study, it was shown that Spa is sufficient for adherence ofbacteria to immobilized VWF under low shear conditions. Recombinant Spaand VWF truncates were used to identify and characterize the domain(s)in each protein that are involved in binding and to refined the VWFbinding domain in SpA by site-directed mutagenesis (O'Seaghdha et al.,2006).

Previous studies have suggested that the SpA-VWF interaction isimportant in S. aureus adherence to platelets under conditions of shearstress and that Spa expression is sufficient for adherence of bacteriato immobilized VWF under low fluid shear (Pawar et al., 2004). Thefull-length recombinant Ig-binding region of SpA, Spa-EDABC, fused toglutathione-S-transferase (GST), bound recombinant VWF in adose-dependent and saturable fashion with half maximal binding of about30 nM in immunosorbent assays. Full-length (FL)-Spa did not bindrecombinant VWF A3 domain but displayed binding to recombinant VWFdomains A1 and D′-D3 (half-maximal binding at 100 nM and 250 nM,respectively). Each recombinant SpA Ig-binding domain bound to the A1domain in a similar manner to the FL-Spa molecule (half-maximal binding100 nM). Amino acid substitutions were introduced in the GST-SpaDprotein at sites known to be involved in IgG Fc or in VH3-Fab binding.Mutants altered in residues that recognized IgG Fc but not those thatrecognized VH3 Fab had reduced binding to VWF-A1 and D′-D3. Thisindicated that both VWF regions recognized a region on helices I and IIthat overlapped the IgG Fc binding site (O'Seaghdha et al., 2006).

Osteomyelitis is a debilitating infectious disease of the bone. It ispredominantly caused by S. aureus and is associated with significantmorbidity and mortality. It is characterized by weakened bonesassociated with progressive bone loss. Currently the mechanism throughwhich either bone loss or bone destruction occurs in osteomyelitispatients is poorly understood (Claro et al., 2011). S. aureus SpA(Protein A) has recently been shown to binds directly to osteoblasts(Claro et al., 2011). This interaction prevents proliferation, inducesapoptosis and inhibits mineralization of cultured osteoblasts. Infectedosteoblasts also increase the expression of RANKL, an important proteininvolved in initiating bone desorption. None of these effects was seenin a mutant of S. aureus lacking SpA. Complementing the SpA-defectivemutant with a plasmid expressing spa or using purified SpA resulted inattachment to osteoblasts, inhibited proliferation and induced apoptosisto a similar extent as wildtype S. aureus. These events demonstratemechanisms through which loss of bone formation and bone weakening mayoccur in osteomyelitis patients.

Staphylococcal SpA is a conserved surface component of all S. aureusstrains, consisting of an N-terminal IgG-binding domain, an Xr or shortsequence-repeat region (SSR) encoded by variable numbers of 24-bprepeated DNA sequences, and a C-terminal anchor to the bacterial cellwall. Resent studies have shown that the Xr domain of SpA, activatesknown components of the type I IFN cascade and that this contributes tothe virulence of the organism as a respiratory pathogen (Martin et al.,2009).

Sbi.

In addition to SpA, many stains of S. aureus also produce Sbi, a secondprotein with Fc binding activity. Sbi is a multidomain protein, whichwas originally identified as an IgG-binding, and β2 glycoprotein-Ibinding protein (Zhang et al., 1998). Sbi is a 436-amino acid proteinthat occurs in many S. aureus strains, including methicillin-sensitive(MSSA) and methicillin-resistant (MRSA) strains. From its N terminus,Sbi includes four small domains up to residue 266 followed by eightcopies of a PXXXX repeat motif with a high concentration of glutamine,lysine, aspartate, valine, and isoleucine and then a C-terminaltyrosine-rich 130-residue region (FIG. 4). Unlike SpA, Sbi lacks thetypical Gram-positive cell wall anchoring sequence LPXTG, but it doeshave a predicted proline-rich cell wall-spanning segment (Zhang et al.,1998). Evidence from Western blots of fractionated cells from several S.aureus strains, including Newman, indicates that most Sbi is secretedinto the medium. It has also been suggested that Sbi is associates withthe bacterial surface through electrostatic interactions (Zhang et al.,1998). Sbi has also been shown to bind a plasma component, adhesionprotein β2-glycoprotein I (β2-GPI), a protein that has been implicatedin blood coagulation (Zhang et al., 1999; Bouma et al., 1999). It hasbeen demonstrated that Sbi interferes directly with the adaptive immunesystem through its two N-terminal IgG binding domains (Sbi-I and Sbi-II)(Zhang, L, 1998), and also modulates the innate immune system throughits third and fourth domains (Sbi-III and Sbi-IV) (Burman et al., 2008).Specifically, Sbi binds complement protein C3 through Sbi-IV interactionwith C3 subunits and induces a futile consumption of complementpredominantly via fluid phase activation of the alternative pathway. Sbifragments containing domains I-II-III-IV (Sbi-E) and III-IV induce thisfutile consumption of complement, whereas isolated Sbi-IV does not.Sbi-IV is nevertheless strongly inhibitory in an assay measuringalternative pathway activation (Burman et al., 2008).

SSL7 and SSL10.

SSL7 (formerly named SET1) and SSL10 are members of the staphylococcalsuperantigen-like (SSL) proteins family (Lina et al., 2004; Williams etal., 2000), related to the staphylococcal enterotoxins (SEs) orsuperantigens. The SSL proteins have 30% sequence identity with toxicshock syndrome 1 (TSST-1) and 25% or less identity with other SEs.Despite the sequence differences, the SSL proteins have a typical SEtertiary structure consisting of a distinctoligonucleotide/oligosaccharide binding (OB-fold) linked to a β-graspdomain (Arcus et al., 2002a; Arcus, 2002b). Similar to the se genes, thessl genes are located in a pathogenicity island (SaPln2) and are likelyto be significant virulence factors. Most healthy individuals haveantibodies to SSL proteins (Al-Shangiti et al., 2005), and the ssl genesexhibit marked allelic variance consistent with selective pressure fromthe host immune system (Baba et al., 2002). However, unlike SE, the SSLproteins do not have superantigen activity, but some have been shown toinhibit important molecules of the host immune system. SSL10(Staphylococcus Super antigen like protein 10) bind IgG1 not IgG2/3/4.The dissociation equilibrium constant for the interaction between humanIgG and recombinant SSL10 was estimated to be 220 nM. Recombinant SSL10inhibited the binding of complement component C1q to IgG. The bindingsite of SSL10 to IgG1 has been located by site directed mutagenesis toresidues within the CH2 domain. Specifically, mutation of IgG1 atresidues 274 and 276 to the residues found in IgG3 (which does not bindSSL 10) abolish binding to the variant IgG1 (Patel et al., 2010). Incontrast to SSL10, SSL7 bind to the Fc domain interface in human IgA.

Antibody Structure and Interactions with Immunoglobulin Binding Proteins

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up ofindividual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins. Each chain is made up of twodistinct regions, referred to as the variable and constant regions. Thelight and heavy chain variable regions show significant sequencediversity between antibodies, and are responsible for binding the targetantigen. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. In humans there are five different classes ofantibodies including IgA (which includes subclasses IgAI and IgA2), IgD,IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), andIgM. The distinguishing features between these antibody classes aretheir constant regions, although subtler differences may exist in theVariable or V region. FIG. 1 shows an IgG antibody, used here as anexample to describe the general structural features of immunoglobulins.IgG antibodies are tetrameric proteins that include two heavy chains andtwo light chains. Each IgG heavy chain includes four immunoglobulindomains linked from N- to C-terminus in the following order: heavy chainvariable domain (VH), heavy chain constant domain 1 (CH1), heavy chainconstant domain 2 (CH2), and heavy chain constant domain 3 (CH3)(VH-CH1-CH2-CH3; also referred to as VH-CγI-Cγ2-Cγ3, referring to theheavy chain variable domain, constant gamma I domain, constant gamma 2domain, and constant gamma 3 domain respectively). The CH1-CH2-CH3 orCγI-Cγ2-Cγ3 domains are also referred to collectively as the heavy chainconstant region. The IgG light chain is composed of two immunoglobulindomains linked from N- to C-terminus in the following order light chainvariable domain (VL) and light chain constant domain (CL) (VL-CL).

Each variable region of an antibody (VH and VL) contains the antigenbinding determinants of the molecule, and thus determines thespecificity of an antibody for its target antigen. The variable regionis so named because it is the most distinct in sequence from otherantibodies within the same class. The majority of sequence variabilityoccurs in the complementarity determining regions (CDRs). There are 6CDRs total, three in each variable domain (VH and VL), designated VHCDRI, VH CDR2, VH CDR3, VL CDRI, VL CDR2, and VL CDR3. The variableregion outside of the CDRs is referred to as the framework (FR) region.Although not as diverse as the CDRs, sequence variability does occur inthe FR region between different antibodies. Overall, this characteristicarchitecture of antibodies provides a stable scaffold (the FR region)upon which substantial antigen binding diversity (the CDRs) can beexplored by the immune system to obtain specificity for a broad array ofantigens.

A number of high-resolution structures are available for a variety ofvariable region fragments from different organisms, some unbound andsome in complex with antigen. The sequence and structural features ofantibody variable regions are well characterized (Morea et al., 1997;Morea et al., 2000), and the conserved features of antibodies haveenabled the development of a wealth of antibody engineering techniques(Maynard et al., 2000). For example, it is possible to graft the CDRsfrom one antibody, for example a murine antibody, onto the frameworkregion of another antibody, for example a human antibody. This process,referred to in the art as “humanization,” enables generation of antibodytherapeutics that have a lower immunogenicity as compared to nonhumanantibodies. Fragments including the variable region can exist in theabsence of other regions of the antibody, including for example, theantigen binding fragment (Fab) which includes VH-CH1 and VH-CL, thevariable fragment (Fv) which includes VH and Vu, the single chainvariable fragment (scFv) which includes VH and VL linked together in thesame chain, as well as a variety of other variable region fragments(Little et al., 2000).

Part of the heavy chain constant region is referred to as the Fc domainor region. The Fc region of an antibody interacts with a number of Fcreceptors and ligands, imparting an array of important functionalcapabilities referred to as effector functions. For IgG the Fc region,as shown in FIG. 1, includes Ig domains CH2 and CH3 and the N-terminalhinge leading into CH2. An important family of Fc receptors for the IgGclass is the Fc gamma receptors (FcγRs). These receptors mediatecommunication between antibodies and the cellular arm of the immunesystem (Raghavan et al., 1996; Ravetch et al., 2001). In humans thisprotein family includes Fcγ RI (CD64), including isoforms FcγRIa,FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R13I), Fcγ RIIb (including Fcγ RIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CDI6), including isoforms FcγRIIa (includingallotypes VI58 and F158) and FcγRIIIb (including allotypes Fcγ RIIIb-NAIand Fcγ RIIIbNA2) (Jefferis et al., 2002). These receptors typicallyhave an extracellular domain that mediates binding to Fc, a membranespanning region, and an intracellular domain that may mediate somesignaling event within the cell. These receptors are expressed in avariety of immune cells including monocytes, macrophages, neutrophils,dendritic cells, eosinophils, mast cells, platelets, B cells, largegranular lymphocytes, Langerhans' cells, natural killer (NK) cells, andγγT cells.

Formation of the Fc/FcγR complex recruits these effector cells to sitesof bound antigen, typically resulting in signaling events within thecells and important subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocytosis,and cytotoxic attack. The ability to mediate cytotoxic and phagocyticeffector functions is a potential mechanism by which antibodies destroytargeted cells. The cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell is referred to as antibodydependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996;Ghetie et al., 2000; Ravetch et al., 2001). The cell-mediated reactionwherein nonspecific cytotoxic cells that express FcγRs recognize boundantibody on a target cell and subsequently cause phagocytosis of thetarget cell is referred to as antibody dependent cell-mediatedphagocytosis (ADCP). In the case of antimicrobial activity, the cellmediated anti-microbial reaction is generally referred to as opsonophagocytosis. Opsonization involves the binding of an opsonin, e.g.,antibody, to a receptor on the pathogen's cell membrane. After opsoninbinds to the membrane, phagocytes are attracted to the pathogen. The Fabportion of the antibody binds to the antigen, whereas the Fc portion ofthe antibody binds to an Fc receptor on the phagocyte, facilitatingphagocytosis. The receptor-opsonin complex can also create byproductslike C3b and C4b which are important components of the complementsystem. These components are deposited on the cell surface of thepathogen and aid in its destruction. A number of structures have beensolved of the extracellular domains of human FcγRs, including FcγRIIa(protein data bank (pdb) accession code IH9V) (Sondermann et al., 2001)(pdb accession code IFCG) (Maxwell et al., 1999), FcγRIIb (pdb accessioncode 2FCB) (Sondermann et al., 1999) and FcγRIIIb (pdb accession codeIE4J) (Sondermann et al., 2000). All FcγRs bind the same region on Fc,at the N-terminal end of the Cγ2 domain and the preceding hinge. Thisinteraction is well characterized structurally (Sondermann et al.,2001), and several structures of the human Fc bound to the extracellulardomain of human FcγRIIIb have been solved (pdb accession code IE4K)(Sondermann et al., 2000), (pdb accession codes HIS and IIIX) (Radaev etal., 2001), as well as has the structure of the human IgE Fc/FcERIacomplex (pdb accession code IF6A) (Garman et al., 2000).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002). All FcγRs bind thesame region on IgG Fc, yet with different affinities: the high affinitybinder FcγRI has a Kd for IgG1 of 10⁻⁸ M whereas the low affinityreceptors FcγRII and FcγRIII generally bind at 10⁻⁶ M and 10⁻⁵ M,respectively. The extracellular domains of FcγRIIa and FcγRIIIb are 96%identical; however FcγRIIIb does not have an intracellular signalingdomain. Furthermore, whereas FcγRI, FcγRIIa/c, and FcγRIIIa are positiveregulators of immune complex-triggered activation, characterized byhaving an intracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM), FcγRIIb has an immunoreceptor tyrosine-basedinhibition motif (ITIM) and is therefore inhibitory. Thus the former arereferred to as activation receptors, and FcγRIIb is referred to as aninhibitory receptor. An overlapping but separate site on Fc, serves asthe interface for the complement protein Clq. Antibodies can alsodestroy pathogens or cancerous cells by complement-dependentcytotoxicity (CDC) whereby antibodies bound to the cell-surface initiatedeposition and activation of early complement components. In the sameway that Fc/FcγR binding mediates opsonophagocytosis, ADCC and ADCP,Fc/Clq binding mediates complement dependent cytotoxicity (CDC) orcomplement deposition on the target cell surface. Clq forms a complexwith the serine proteases Clr and Cls to form the Cl complex. Clq iscapable of binding six antibodies, although binding to two IgGs issufficient to activate the complement cascade. Similar to Fc interactionwith FcγRs, different IgG subclasses have different affinity for Clq,with IgG1 and IgG3 typically binding substantially better to the FcγRsthan IgG2 and IgG4 (Jefferis et al., 2002). There is currently nostructure available for the Fc/Clq complex; however, mutagenesis studieshave mapped the binding site on human IgG for Clq to a region involvingresidues D270, K322, K326, P329, and P331, and E333 (Idusogie et al.,2000; Idusogie et al., 2001).

A site on Fc between the CH2 and CH3 domains of IgG, mediatesinteraction with the neonatal receptor FcRn, the binding of whichrecycles endocytosed antibody from the endosome back to the bloodstream(Raghavan et al., 1996; Ghetie et al., 2000). This process, coupled withpreclusion of kidney filtration due to the large size of the full-lengthmolecule, results in favorable antibody serum half-lives ranging fromone to three weeks. Binding of Fc to FcRn also plays an important rolein antibody transport.

The binding site for FcRn on Fc overlaps with the site at which S.aureus SpA, streptococcal Protein G and a variety of other microbial FcBinding Proteins (FcBP) bind. The tight binding by these proteins hasbeen exploited as a means to purify antibodies by employing SpA orProtein G affinity chromatography during protein purification. Thus, thefidelity of this region on Fc is important for both the clinicalproperties of antibodies and their purification. Available structures ofthe rat Fc/FcRn complex (Martin et al., 2001), and of the complexes ofFc with Proteins A and G (Deisenhofer, 1981; Sauer-Eriksson et al.,1995; Tashiro et al., 1995) provide insight into the interaction of Fcwith these proteins. An important feature of the Fc region is theconserved N-linked glycosylation that occurs at N297, shown in FIG. 1.This carbohydrate, or oligosaccharide as it is sometimes referred, playsan important structural and functional role for the antibody, and is oneof the principle reasons that antibodies must be produced usingmammalian expression systems. While not wanting to be limited to onetheory, it is believed that the structural purpose of this carbohydratemay be to stabilize or solubilize Fc, determine a specific angle orlevel of flexibility between the Cγ3 and Cγ2 domains, keep the two Cγ2domains from aggregating with one another across the central axis, or acombination of these. Efficient Fc binding to FcγR and Clq requires thismodification and alterations in the composition of the N297 carbohydrateor its elimination affect binding to these proteins (Umaiia et al.,1999; Davies et al., 2001; Mimura et al., 2001; Radaev et al., 2001;Shields et al., 2001; Shields et al., 2002; Simmons et al., 2002). Yetthe carbohydrate makes little if any specific contact with FcγRs (Radaevet al., 2001), indicating that the functional role of the N297carbohydrate in mediating Fc/Fcγ R binding may be via the structuralrole it plays in determining the Fc conformation. This is supported by acollection of crystal structures of four different Fc glycoforms, whichshow that the composition of the oligosaccharide impacts theconformation of Cγ2 and as a result the Fc/Fcγ R interface (Krapp etal., 2003).

The features of antibodies discussed above-specificity for its target,ability to mediate immune effector functions, and good half-lifes inserum-make antibodies powerful therapeutics. Monoclonal antibodies areused therapeutically for the treatment of a variety of conditionsincluding cancer, inflammation, cardiovascular disease and infectiousdiseases. There are currently several antibody products on the marketand hundreds in development. In addition to antibodies, an antibody-likeprotein that is finding an expanding role in research and therapy is theFc fusion (Chamow et al., 1996; Ashkenazi et al., 1997). An Fc fusion isa protein wherein one or more polypeptides are operably linked to Fc. AnFc fusion combines the Fc region of an antibody, and thus its favorableeffector function and pharmacokinetics, with the target-binding regionof a receptor, ligand, or some other protein or protein domain. The roleof the latter is to mediate target recognition, and thus it isfunctionally analogous to the antibody variable region. Because of thestructural and functional overlap of Fc fusions with antibodies, thediscussion on antibodies herein is also applicable to Fc fusions.

The mechanisms by which an antibody neutralizes pathogenic material canbe diverse, including antibody dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis, complement-dependentcytotoxicity (CDC), opsonization, and steric hindrance, almost all ofwhich require the antibody Fc region to interact with cellular receptors(Marasco & Sui, 2007; Lehner 1989; Lazar et al. 2006). For instance,ADCC depends upon the Fc interaction with the activating FcγRIIIareceptor, present on natural killer cells and other leukocytes.Increasing the affinity and selectivity of this interaction throughthree Fc amino acid substitutions increased ADCC by two orders ofmagnitude in vitro (Lazar et al., U.S. Patent Publication No.20080242845). Additionally, heavy chain constant region variants withincreased ability to recruit complement have been described. Variantsdemonstrated enhanced potency in a cell-based CDC assay and improvedbinding affinity to C1q. (Moore et al., 2010)

Antibodies have been used to bind and inactivate pathogenic material formany years, originally being isolated as polyclonal antibody mixturesfrom immunized horse serum. This “passive immunotherapy” was usedsuccessfully to treat many viral and bacterial infections but due tonumerous problems, including product heterogeneity and low specifictiter, coupled with risks of immunogenicity and viral contamination,lost favor after the introduction of antibiotics (Casadevall et al.,2004).

The emergence of antibiotic-resistant microorganisms, emerging virusesand the threat of engineered microorganisms coupled with advances inunderstanding pathogenic mechanisms and antibody technology leaves thisclass of therapeutics poised for a comeback (Casadevall et al., 2004;Zeitlin et al., 2000). Antibodies are attractive anti-infectivetherapeutics for their ability to recognize pathogen-associated ligandmolecules with exquisite specificity and to recruit additional immunesystem components such as complement and natural killer cells,facilitating pathogen inactivation and removal. When properly designed,an antibody can effectively eliminate or control the infection.Unfortunately, efforts to develop recombinant monoclonal antibodies thatrecapitulate polyclonal anti-sera has not been straightforward, largelydue to challenges in identifying appropriate target epitopes, microbialevasion of the humeral immune response and interactions with the rest ofthe immune system. In the cases of RSV and anthrax, importantneutralizing epitopes have been identified, resulting in a remarkablysuccessful drug in the first case and several promising candidates inthe second. Several approaches to treating infection involve antibodiesthat directly bind surface-exposed or associated molecules on wholepathogen cells. These antibodies (depending on the isotype selected) canact by (1) recruiting immune system components to eliminate the pathogenthrough antibody effector functionalities (e.g., complement, CDC, ADCC,ADCP and opsonophagocytosis); (2) blocking cell associated pathogenicmechanisms, i.e., type III secretion of virulence factors; and (3)directly killing pathogens by targeted delivery of chemotherapeuticagents. These approaches are less developed than those for antiviral oranti-toxin therapies in that none have been approved for use and severalpromising candidates for treatment of Staphylococcus infections reachedPhase III trials, only to miss their efficacy targets.

Variant Immunoglobulins Having Attenuated Binding to Virulence Factors

According to the embodiments described herein, variant antibodies havingattenuated non-immune binding to one or more IgBP virulence factors areprovided. In some embodiments, the antibodies have a variant heavy chainconstant region (i.e., a variant CH1-CH2-CH3 domain or variantCγI-Cγ2-Cγ3 domain) having attenuated binding to one or more microbialimmunoglobulin binding proteins (IgBPs).

According to the embodiments described herein, the disclosure providesanti-microbial monoclonal immunoglobulins, such as variant IgGimmunoglobulins, in which at least one amino acid from the IgG heavychain constant region is substituted with another amino acid which isdifferent from that present in the parent antibody. The amino acidsubstitution or substitutions may be in any one or more of the heavychain constant domains, CH1, CH2 or CH3.

In some embodiments, the monoclonal antibody is a mammalian, chimeric,humanized of human anti-microbial IgG variant antibody in which at leastone amino acid from an IgG heavy chain constant region, is substitutedwith at least one amino acid that differs from that present in theparent antibody. Such variant anti-microbial antibodies have attenuatedFc binding to one or more microbial Fc Binding proteins or Fc bindingprotein domains expressed by the target microbe.

In other embodiments, the monoclonal antibody is a animal anti-microbialIg variant antibody for veterinary use, in which at least one amino acidfrom the IgG heavy chain constant region is substituted with anotheramino acid which is different from that present in the parent antibody.Such variant anti-microbial antibodies have attenuated Fc binding to oneor more microbial Fc Binding proteins or Fc binding protein domainsexpressed by the target microbe.

The variant immunoglobulin IgG heavy chain and light chain constantregions described herein can be combined with immunoglobulin variableheavy and light chain regions (the variable domain), which bind antigensproduced by microbes that express one or more microbial immunoglobulinbinding protein.

In some embodiments, the variable domain of the antibody binds to amicrobial protein that is a microbial immunoglobulin binding protein,and the heavy chain constant regions of the antibody is a variant IgG Fcwhich has attenuated binding to one or more microbial Ig Binding Proteinor Fc Binding Protein domain expressed by the target microbe.

In other embodiments, the variable domain of the antibody binds to amicrobial protein that is not an microbial immunoglobulin bindingprotein, and the heavy chain constant region of the antibody is avariant IgG Fc which has attenuated binding to one or more microbial IgBinding Proteins or Fc Binding Protein domains expressed by the targetmicrobe.

The heavy chain constant region variant IgG immunoglobulins claimedherein have enhanced antimicrobial activity relative to their parentalantibodies.

In some embodiments such variant IgG heavy chain constant polypeptidesequences are combined with immunoglobulin heavy chain variablepolypeptide sequences and light chains polypeptide sequences, which bindone or more cell surface or secreted microbial antigen.

In some embodiments, immunoglobulins with variant heavy chain constantregions having altered (i.e., decreased) non-immune binding to one ormore microbial IgBP are provided. The embodiments described hereinprovide modified antibodies having altered non-immune IgBP bindingrelative to the corresponding unmodified antibody. More particularly,the embodiments described herein are directed to variant human orhumanized monoclonal antibodies directed against microbial surfaceantigens or surface associated antigens, which have attenuated Fcbinding to one or more microbial IgBPs.

The embodiments described herein are directed to variant IgGimmunoglobulin heavy chain constant region-containing polypeptides thathave attenuated heavy chain constant regions binding to one or moremicrobial IgBPs as a consequence of the introduction of amino acidchanges within the immunoglobulin heavy chain region.

According to the embodiments described herein, the variantanti-microbial antibodies of the disclosure may include one or moresequences derived from at least 4 regions of the IgG antibody. Theseregions include, but are not limited to:

-   -   The heavy chain constant region, which includes domains CH1, the        hinge region, CH2 and CH3. This region of the antibody is        responsible for the effector function of the antibody. In some        embodiments, this region is derived from human IgG1. In        alternative embodiments, the Fc region is of mixed isotype in        which the CH3 domain of IgG1, or the CH2 and CH3 domains of        IgG1, are exchanged with their homologous domains from IgG3 of        any human allotype. The EU numbering of the heavy chain constant        region corresponds to approximate positions of H118-H446    -   The heavy chain variable domain, which contains the antigen        recognition region of the heavy chain, including the CDR1, CDR2        and CDR3 and framework regions. This region can be derived from        a human antibody, from a chimeric or humanized antibody, or by        humanization of a non-human antibody.    -   The light chain constant regions: In one embodiment, the light        chain constant region is a kappa light chain. In other        embodiments the light chain constant region is a lambda light        chain. The EU numbering positions for a light chain correspond        to approximate positions of L108-L214    -   The light chain variable domain, which includes the antigen        recognition region of the light chain, including the CDR1, CDR2        and CDR3 and framework regions. This region can be derived from        a human antibody, from a chimeric or humanized antibody, or by        humanization of a non-human antibody.

In some embodiments, the heavy chain constant region variant antibody isof IgG immunoglobulin, in which at least one amino acid from the heavychain constant region selected from, but not limited to amino acidresidues (i.e., EU positions) 214, 251, 252, 253, 254, 274, 276, 311,314, 356, 358, 380, 382, 384, 419, 422, 428, 431, 432, 433, 434, 435,436 and 438 is substituted with an amino acid residue different fromthat present in the unmodified IgG1 antibody. The substitutionsdescribed herein are not limiting and in some aspect, additionalsubstitution residues may be made. Further, at least one amino acid fromthe heavy chain constant region may be a single amino acid substitutionalone, or a combination of at least two amino acids selected from anycombination of one or more of the amino acid substitutions describedherein, combined with one or more second amino acid substitutiondescribe herein, or alternatively, may be combined with anothersubstitution not disclosed herein. Substitutions, either alone or incombinations, attenuates the binding of one or more microbial Ig BindingProtein to the heavy chain constant region of the variant antibody

In some embodiments, the heavy chain constant region variant antibody isof isotype IgG1, in which at least one amino acid from the heavy chainconstant region selected from, but not limited to amino acid residues(i.e., EU positions) 214, 251, 252, 253, 254, 274, 276, 311, 314, 356,358, 380, 382, 384, 419, 422, 428, 431, 432, 433, 434, 435, 436 and 438is substituted with an amino acid residue different from that present inthe unmodified IgG1 antibody. The substitutions described herein are notlimiting and in some aspect, additional substitution residues may bemade. Further, at least one amino acid from the heavy chain constantregion may be a single amino acid substitution alone, or a combinationof at least two amino acids selected from any combination of one or moreof the amino acid substitutions described herein, combined with one ormore second amino acid substitution describe herein, or alternatively,may be combined with another substitution not disclosed herein.Substitutions, either alone or in combinations, attenuates the bindingof one or more microbial Ig Binding Protein to the heavy chain constantregion of the variant antibody

In embodiments in which the heavy chain constant region variantanti-microbial antibody is directed against S. aureus, amino acidchanges can be introduced into the heavy chain constant CH2 domain toattenuate SSL10 binding to the variant immunoglobulin. Separatemutations can be introduced into the CH2 or CH3 domain to attenuate Sbiand/or SpA binding to the Fc domain. Mutations can also be introducedinto the heavy chain variable FW region to attenuate superantigen typeSpA binding to the Fab domain of VH3 derived antibodies.

In some embodiments, amino acid residue (i.e., EU position) 435 from theheavy chain constant region is substituted with Arg, resulting inattenuated binding of the Fc domain of the variant antibody to Sbiand/or SpA (Including but not limited to SEQ ID: 31-46).

In some embodiments, amino acid residue (i.e., EU position) 435 from theheavy chain constant region is substituted with Arg and amino acidresidue (i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln resulting in attenuated binding of the Fc domain ofthe variant antibody to Sbi and SSL 10 or SpA (Including but not limitedto SEQ ID: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56)

In some embodiments, amino acid residue (i.e., EU position) 435 from theheavy chain constant region is substituted with Arg and amino acidresidue (i.e., EU position) 436 from the heavy chain constant region issubstituted with Phe resulting in attenuated binding of the Fc domain ofthe variant antibody to Sbi and/or SpA (Including but not limited to SEQID: 39-46).

In some embodiments, amino acid residue (i.e., EU position) 435 from theheavy chain constant region is substituted with Arg, amino acid residue(i.e., EU position) 436 from the heavy chain constant region issubstituted with Phe and amino acid residue (i.e., EU position) 274 fromthe heavy chain constant region is substituted with Gln resulting inattenuated binding of the Fc domain of the variant antibody to Sbi andSSL 10 and/or SpA (Including but not limited to SEQ ID: 40, 42, 44, 46)

In some embodiments, amino acid residue (i.e., EU position) 422 from theheavy chain constant region is substituted with Ile and amino acidresidue (i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg resulting in attenuated binding of the variantantibody to Sbi and/or SpA (Including but not limited to SEQ ID 33, 34,37, 38, 41, 42, 45, 46).

In some embodiments, amino acid residue (i.e., EU position) 422 from theheavy chain constant region is substituted with Ile, amino acid residue(i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln and amino acid residue (i.e., EU position) 435 fromthe heavy chain constant region is substituted with Arg resulting inattenuated binding of the variant antibody to Sbi and SSL 10 and/or SpA(Including but not limited to SEQ ID 34, 38, 42, 46).

In some embodiments, amino acid residue (i.e., EU position) 422 from theheavy chain constant region is substituted with Ile, amino acid residue(i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg and amino acid residue (i.e., EU position) 436 fromthe heavy chain constant region is substituted with Phe resulting inattenuated binding of the variant antibody to Sbi and/or SpA (Includingbut not limited to SEQ ID: 41, 42, 45, 46).

In some embodiments, amino acid residue (i.e., EU position) 422 from theheavy chain constant region is substituted with Ile, amino acid residue(i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln, amino acid residue (i.e., EU position) 435 fromthe heavy chain constant region is substituted with Arg and amino acidresidue (i.e., EU position) 436 from the heavy chain constant region issubstituted with Phe resulting in attenuated binding of the variantantibody to Sbi and/or SpA (Including but not limited to SEQ ID:42, 46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu and amino acidresidue (i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg resulting in attenuated binding of the variantantibody to Sbi and/or SpA (Including but not limited to SEQ ID: 35-38,43-46)

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg and amino acid residue (i.e., EU position) 274 fromthe heavy chain constant region is substituted with Gln resulting inattenuated binding of the variant antibody to Sbi and SSL10 and/or SpA(Including but not limited to SEQ ID: 36, 38 44, 46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg and amino acid residue (i.e., EU position) 436 fromthe heavy chain constant region is substituted with Phe, resulting inattenuated binding of the variant antibody to Sbi and/or SpA (Includingbut not limited to SEQ ID: 43-46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg, amino acid residue (i.e., EU position) 274 fromthe heavy chain constant region is substituted with Gln and amino acidresidue (i.e., EU position) 436 from the heavy chain constant region issubstituted with Phe, resulting in attenuated binding of the variantantibody to Sbi and/or SpA (Including but not limited to SEQ ID: 44,46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 422 from the heavy chain constant region issubstituted with Ile and amino acid residue (i.e., EU position) 435 fromthe heavy chain constant region is substituted with Arg resulting inattenuated binding of the variant antibody to Sbi and/or SpA (Includingbut not limited to SEQ ID: 37, 38, 45, 46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 422 from the heavy chain constant region issubstituted with Ile, amino acid residue (i.e., EU position) 274 fromthe heavy chain constant region is substituted with Gln and amino acidresidue (i.e., EU position) 435 from the heavy chain constant region issubstituted with Arg resulting in attenuated binding of the variantantibody to Sbi and SSL10 and/or SpA (Including but not limited to SEQID: 38, 46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 422 from the heavy chain constant region issubstituted with Ile, amino acid residue (i.e., EU position) 435 fromthe heavy chain constant region is substituted with Arg and amino acidresidue (i.e., EU position) 436 from the heavy chain constant region issubstituted with Phe, resulting in attenuated binding of the variantantibody to Sbi and/or SpA (Including but not limited to SEQ ID: 45,46).

In some embodiments, amino acid residue (i.e., EU position) 419 from theheavy chain constant region is substituted with Glu, amino acid residue(i.e., EU position) 422 from the heavy chain constant region issubstituted with Ile, amino acid residue (i.e., EU position) 435 fromthe heavy chain constant region is substituted with Arg, amino acidresidue (i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln and amino acid residue (i.e., EU position) 436 fromthe heavy chain constant region is substituted with Phe, resulting inattenuated binding of the variant antibody to Sbi and SSL10 and/or SpA(Including but not limited to SEQ ID: 46).

In some embodiments, amino acid residue (i.e., EU position) 436 from theheavy chain constant region is substituted with Phe resulting inattenuated binding of the Fc domain of the variant antibody to Sbiand/or SpA (Including but not limited to SEQ ID: 39-48, 53-56).

In some embodiments, amino acid residue (i.e., EU position) 436 from theheavy chain constant region is substituted with Phe and amino acidresidue (i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln resulting in attenuated binding of the Fc domain ofthe variant antibody to Sbi and SSL 10 and/or SpA (Including but notlimited to SEQ ID: 40, 42, 44, 46, 48, 54, 56).

In some embodiments, amino acid residue (i.e., EU position) 254 from theheavy chain constant region is substituted with Thr resulting inattenuated binding of the variant antibody to Sbi and/or SpA (Includingbut not limited to SEQ ID: 49-56).

In some embodiments, amino acid residue (i.e., EU position) 254 from theheavy chain constant region is substituted with Thr and amino acidresidue (i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln resulting in attenuated binding of the variantantibody to Sbi and SSL10 and/or SpA (Including but not limited to SEQID: 50, 52, 54, 56).

In some embodiments, amino acid residue (i.e., EU position) 252 and 254from the heavy chain constant region are substituted with Thr resultingin attenuated binding of the variant antibody to Sbi and/or SpA(Including but not limited to SEQ ID: 51, 52, 55, 56)

In some embodiments, amino acid residue (i.e., EU position) 252 and 254from the heavy chain constant region are substituted with Thr, and aminoacid residue (i.e., EU position) 274 from the heavy chain constantregion is substituted with Gln resulting in attenuated binding of thevariant antibody to Sbi and SSL10 and/or SpA (Including but not limitedto SEQ ID: 52, 56)

In some embodiments, amino acid residue (i.e., EU position) 254 from theheavy chain constant region is substituted with Thr and amino acidresidue (i.e., EU position) 456 from the heavy chain constant region issubstituted with Phe resulting in attenuated binding of the variantantibody to Sbi and/or SpA (Including but not limited to SEQ ID: 53-56).

In some embodiments, amino acid residue (i.e., EU position) 254 from theheavy chain constant region is substituted with Thr, amino acid residue(i.e., EU position) 274 from the heavy chain constant region issubstituted with Gln and amino acid residue (i.e., EU position) 456 fromthe heavy chain constant region is substituted with Phe resulting inattenuated binding of the variant antibody to SSL10 and Sbi and/or SpA(Including but not limited to SEQ ID: 54, 56).

In some embodiments, amino acid residue (i.e., EU position) 252 and 254from the heavy chain constant region are substituted with Thr and aminoacid residue (i.e., EU position) 456 from the heavy chain constantregion is substituted with Phe resulting in attenuated binding of thevariant antibody to Sbi and/or SpA (Including but not limited to SEQ ID:55, 56).

In some embodiments, amino acid residue (i.e., EU position) 252 and 254from the heavy chain constant region are substituted with Thr, aminoacid residue (i.e., EU position) 456 from the heavy chain constantregion is substituted with Phe and amino acid residue (i.e., EUposition) 274 from the heavy chain constant region is substituted withGln resulting in attenuated binding of the variant antibody to Sbi andSSL10 and/or SpA (Including but not limited to SEQ ID: 56).

In some embodiments, amino acid residue (i.e., EU position) 274 and/or276 from the heavy chain constant region are substituted with anotheramino acid, which is different from that present in an unmodifiedparental antibody. The resulting variant antibody variant antibody hasattenuated binding to the SSL10 IgBPs compared with the unmodifiedantibody.

In some embodiments, amino acid residue (i.e., EU position) 274 from theheavy chain constant region is substituted with Gln, which is differentfrom that present in an unmodified parental antibody. The resultingvariant antibody has attenuated binding to SSL10 IgBPs compared with theunmodified antibody (Including but not limited to SEQ ID: 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56).

In some embodiments, amino acid residue (i.e., EU position) 276 from theheavy chain constant region is substituted with Lys, which is differentfrom that present in an unmodified parental antibody. The resultingvariant antibody has attenuated binding to SSL10 IgBPs compared with theunmodified antibody.

In some embodiments, amino acid residue (i.e., EU position) 274 and 276from the heavy chain constant region is substituted with Gln and Lysrespectively, which is different from the residues present in anunmodified parental antibody. The resulting variant antibody hasattenuated binding affinity for SSL10 IgBPs compared with the unmodifiedantibody.

In some embodiments, where the immunoglobulin is directed against astaphylococcal antigen, a variant IgG1 antibody having attenuatedbinding to S. aureus SSL 10 as compared with the parental antibody isprovided, wherein either one of both of amino acid residues (i.e., EUposition) 274 and 276 from the heavy chain constant region aresubstituted with Gln and Lys respectively.

In some embodiments, antibodies having a heavy chain constant regionsubstantially identical to a naturally occurring class IgG1 antibodyconstant region are provided, wherein at least two amino acid residueselected from residues (i.e., EU positions) 214, 251, 252, 253, 254,274, 276, 311, 314, 356, 358, 380, 382, 384, 419, 422, 428, 431, 432,433, 434, 435, 436 and 438 are different from that present in theparental antibody, thereby attenuating binding of the variant heavychain constant region (relative to the parental antibody) to one or moreFcBP selected from the list: S. aureus SSL10, Sbi and Protein.

In some embodiments, antibodies having a heavy chain constant regionsubstantially identical to a naturally occurring class IgG1 antibodyheavy chain constant region are provided, wherein at least three aminoacid residue selected from residues (i.e., EU positions) 214, 251, 252,253, 254, 274, 276, 311, 314, 356, 358, 380, 382, 384, 419, 422, 428,431, 432, 433, 434, 435, 436 and 438 are different from that present inthe naturally occurring class IgG1 antibody, thereby attenuating bindingof the variant heavy chain constant region (relative to the parentalantibody) to one or more FcBP selected from the list: S. aureus SSL10,Sbi and Protein.

In some embodiments, antibodies having a heavy chain constant regionsubstantially identical to a naturally occurring class IgG1 antibodyheavy chain constant region are provided, wherein at least three aminoacid residue selected from residues (i.e., EU positions) 274, 276, 419,422, 435 and 436 are different from that present in the naturallyoccurring class IgG1 antibody, thereby attenuating binding of thevariant heavy chain constant region (relative to the parental antibody)to one or more FcBP selected from the list: S. aureus SSL10, Sbi andProtein.

In some embodiments, antibodies having a heavy chain constant regionsubstantially identical to a naturally occurring class IgG1 antibodyconstant region are provided, wherein at least three amino acid residueselected from residues (i.e., EU positions) 214, 252, 254, 274, 276,356, 358, 419, 422, 431, 435 and 436 are different from that present inthe parental antibody, thereby attenuating binding of the variant heavychain constant region (relative to the parental antibody) to one or moreFcBP selected from the list: S. aureus SSL10, Sbi and Protein.

In some embodiments, allotypic versions of variant IgG1 antibodies withattenuated microbial FcBP binding to the variant Fc domain of theantibody are provided, wherein at least one heavy chain amino acidresidue selected from residues (i.e., EU positions) 214, 356, 358 and431 of the heavy chain are different from that present in the parentalantibody.

In some embodiments, iso-allotypic versions of variant IgG1 antibodieswith attenuated FcBP binding to the variant heavy chain constant regionof the antibody are claimed, wherein at least one heavy chain amino acidresidue selected from residues (i.e., EU positions) 214, 251, 252, 253,254, 274, 276, 311, 314, 356, 358, 380, 382, 384, 419, 422, 428, 431,432, 433, 434, 435, 436 and 438 of the heavy chain are different fromthat present in the parental antibody.

In some embodiments, iso-allotypic version of variant IgG1 antibodieswith attenuated microbial FcBP binding to the variant heavy chainconstant region of the antibody are claimed, wherein at least one heavychain amino acid residue selected from residues (i.e., EU positions)214, 251, 252, 253, 254, 274, 276, 311, 314, 356, 358, 380, 382, 384,419, 422, 428, 431, 432, 433, 434, 435, 436 and 438 of the heavy chainare different from that present in the parental antibody. In embodimentsamino acid 365 is Glu, 358 is Met, 431 is Ala and 214 is Lys.

In some embodiments, the heavy chain constant region of the variant IgG1antibody has decreased binding to one or more microbial FcBPs selectedfrom the list including, but not limited to S. aureus Sbi, SpA and SSL10compared with the parental antibody, in which at least two heavy chainconstant region amino acids selected from residues (i.e., EU positions)214, 251, 252, 253, 254, 274, 276, 311, 314, 356, 358, 380, 382, 384,419, 422, 428, 431, 432, 433, 434, 435, 436 and 438 are substituted withamino acid residues different from that present in the parental IgG1antibody.

In some embodiments the heavy chain constant region of the variant IgG1antibody has decreased binding to one or more microbial FcBPs selectedfrom the group including, but not limited to S. aureus Sbi, SpA andSSL10 compared with the parental antibody, in which at least three heavychain constant region amino acids selected from residues (i.e., EUpositions) 214, 251, 252, 253, 254, 274, 276, 311, 314, 356, 358, 380,382, 384, 419, 422, 428, 431, 432, 433, 434, 435, 436 and 438 aresubstituted with amino acid residues different from that present in theparental IgG1 antibody.

In some embodiments, the heavy chain constant region of the antibody, orvariant antibody, contains a heavy chain constant region of isotypeG1m17.

In some embodiments, the heavy chain constant region of the antibody, orvariant antibody, contains a heavy chain constant region of isotypeG1m17 that includes an amino acid sequences selected from the groupheavy chain constant region 1-27 (SEQ ID NO:30-56).

In other embodiments, the heavy chain constant region of the antibody,or variant antibody, may be substantially encoded by any allotype orisoallotype of any immunoglobulin gene. In one embodiment, the heavychain constant region variants comprise IgG1 sequences that areclassified as Glm(1), Glm(2), Glm(3), Glm(17), nGlm(I), nGlm(2), and/ornGlm(17). Thus, in the context of an IgG1 isotype, the heavy chainconstant region variants may comprise a Lys (Glm(17)) or Arg (Glm(3)) atposition 214, an Asp356/Leu358 (Glm(1)) or Glu356/Met358 (nGlm(1),and/or a Gly (Glm(2)) or Ala (nGlm(2)) at position 431.

In an alternative embodiment, the antibody variant has a constant heavychain region of mixed isotype, created by substituting the CH2 and CH3domains of the parental IgG1 heavy chain constant region with the CH2and CH3 domains from the IgG3 heavy chain contain region. In someembodiments, the IgG3 heavy chain sequences can be from IgG3 allotypesG3m5,10,11,13,14, G3m5,6,10,11,14, G3m5,6,11,24 or G3m21,28.

In an alternative embodiment, the antibody variant has a constant heavychain region of mixed isotype, created by substituting the CH3 domainsof the parental IgG1 heavy chain constant region with the CH3 domainsfrom IgG3 heavy chain contain region. In some embodiments, the IgG3heavy chain sequences can be from IgG3 allotypes G3m5,10,11,13,14,G3m5,6,10,11,14, G3m5,6,11,24 or G3m21,28.

In some embodiments, the variant antibodies are of mixed isotype,wherein the IgG1/IgG3 fusion junction is located between amino acidresidues (i.e., EU position) 236 and 237.

In some embodiments, the variant antibodies are of mixed isotype,wherein the IgG1/IgG3 fusion junction is located between amino acidresidues (i.e., EU position) 340 and 341.

In some embodiments, variant antibodies of mixed isotype having theIgG1/IgG3 fusion junction located between amino acid residues (i.e., EUposition) 236 and 237, have one or more amino acid from the mixedisotype heavy chain constant region selected from amino acid residues(i.e., EU position) 214, 251, 252, 253, 254, 274, 276, 311, 314, 356,358, 380, 382, 384, 419, 422, 428, 431, 432, 433, 434, 435, 436 and 438that is substituted with an amino acid residue different from thatpresent in the parental mixed isotype antibody.

In some embodiments, variant antibodies of mixed isotype having theIgG1/IgG3 fusion junction located between amino acid residues (i.e., EUposition) 340 and 341, have one or more amino acid from the mixedisotype heavy chain constant region selected from amino acid residues(i.e., EU position) 214, 251, 252, 253, 254, 274, 276, 311, 314, 356,358, 380, 382, 384, 419, 422, 428, 431, 432, 433, 434, 435, 436 and 438,that is substituted with an amino acid residue different from thatpresent in the parental mixed isotype antibody.

In some embodiments, the antibodies described herein may have a variantheavy chain variable region having attenuated non-immune binding to oneor more S. aureus superantigens such that the antibody has low or nosuperantigen type binding to SpA. Such immunoglobulins and theirvariants can be selected so as to avoid the use of human VH3 derivedsequences, which can interact with SpA at a site distinct from the Fcbinding site. Alternatively, if VH3 derived sequences are used, andFab-SpA superantigen type binding is present in the parentalimmunoglobulin, then modified variable heavy chains are provided inwhich at least one amino acid from the heavy chain variable region issubstituted with an amino acid residue different from that present inthe unmodified parental antibody selected from the list of VH residuesincluding but not limited to H15, H17, H19, H57, H59, H64, H65, H66,H68, H69, H70, H80, H81 and H82 (including H82a and other H82 positions)numbered according to Kabat. In some aspects, VH region variants mayreduce or abolish the superantigen type binding of the Fab region ofsaid variant antibody to S. aureus SpA relative to the parentalantibody, but do not significantly attenuate antigen binding to theantigen binding site of the variant antibody.

In certain embodiments, the antibody has a variant Fab region thatattenuates non-immune binding to an S. aureus superantigen such as SpA,and also has one or more heavy chain constant region substitutions thatattenuate Fc binding with one or more S. aureus FcBPs. In suchembodiments, the antimicrobial antibody, or variant antibody, contains aheavy chain constant region selected from heavy chain constant regions1-27 (SEQ ID NO: 30-56), and a heavy chain variable domain in which atleast one amino acid selected from the list of VH3 residues includingH15, H17, H19, H57, H59, H64, H65, H66, H68, H69, H70, H80, H81 and H82(including H82a and other H82 positions according to Kabat numbering) issubstituted with an amino acid residues different from that present inthe parental IgG1 antibody.

In some embodiments, the antimicrobial variant IgG1 heavy chain ispaired with a kappa light chain of allotype selected from the group Km1,Km2, Km3.

In some embodiments, the antimicrobial variant IgG1 heavy chain ispaired with a lambda light chain.

In some embodiments, the antimicrobial variant IgG1 heavy chain ispaired with a kappa light chain having either amino acid Val or Ala atposition 153 and/or either Leu or Val at amino acid 191 (EU numbering).

To compare the effect of variant heavy chain constant region changes onthe binding and effector properties of anti microbial IgGimmunoglobulins, control antibodies including parental IgGimmunoglobulins and a humanized anti RSV antibody having a matchedvariant heavy chain constant region are produced and tested. Suchcontrols are important in distinguishing antigen binding by the variabledomain of the antibody from heavy chain constant region binding to thetarget microbial antigen or microbe.

Additional Embodiments of Claimed Heavy Chain Constant Region VariantImmunoglobulins

In some embodiments, the variant immunoglobulins of the presentdisclosure have enhanced antimicrobial effector function. According tothe embodiments described herein, the enhanced anti-microbial effectorfunction, may include, but is not limited to, C1q binding, C3bdeposition, ADCC, ADCP, CDC, opsonophagocytic activity, antimicrobialactivity, or a combination thereof.

In some embodiments, the variant immunoglobulins of the presentdisclosure may have altered microbial FcBP and FcRn binding to the heavychain constant region, without significantly altering other antibodyeffector functions such as C1q binding or Fc gamma receptor binding tothe variant Fc domain.

The heavy chain constant region variant immunoglobulins of the presentdisclosure may be combined with other Fc modifications known in the art(e.g. Shields el al., J. Biol. Chem, 2001, 276, 6591-6604; Dall'Acqua etal., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 33, pp.23514-23524, Aug. 18, 2006; reviewed in Natsume et al., Drug Design,Development and Therapy 2009:3 7-16, which are hereby incorporated byreference as if fully set forth herein). The embodiments describedherein encompass combining an immunoglobulin or variant thereof, such asthose described herein, with other known constant domain modificationsto provide additive, synergistic, or novel properties to the modifiedantibody. The modifications known in the art may enhance the phenotype(anti-microbial activity) of the immunoglobulin or variantimmunoglobulins with which they are combined. For example, an IgG Fcdomain variant described herein with reduced Fc binding to S. aureusSpA, SSL 10 or Sbi may be combined with one or more heavy chain constantregion mutations known to result in C1q binding with higher affinitythan a comparable wild type constant region. Such claimed embodimentsresults in enhanced antimicrobial effector function.

Additionally, mutation or alternations to the hinge region of thevariant heavy chain constant region, which enhances stability or thevariant immunoglobulin with respect to microbial protease cleavage, arealso claimed. Examples of such microbial proteases include but are notlimited to IdeS, GluV8 and SpeB.

Some embodiments described herein also relate to modified variant IgGimmunoglobulins that have decreased in vivo half-life by virtue of thepresence of a modified human IgG1 heavy chain constant region, whereinthe IgG heavy chain constant region, or fragment thereof, is modified bythe introduction of one or more amino acid changes. The one or moreamino acid changes may be an amino acid substitution, or by theengineering of a mixed isotype IgG constant domains, all of which havedecreased affinity for one or more microbial FcBP and for the human FcRnreceptor.

In some embodiments, modified variant class IgG1 antibodies areprovided, wherein the in vivo half-lives are reduced by changes in oneor more amino acid residues at positions which have been identified tobe involved, either directly or indirectly, in the interaction of theIgG1 with the FcRn receptor. The altered half-life resulting fromreduced FcRn binding will decrease the half-life of the modified variantIgG1 relative to a parental IgG1 molecule. This altered half-life willallow better control of patient exposure in the clinic.

In further embodiments, methods for modifying an antibody of class IgG1or mixed isotype are provided, wherein said method includes substitutingat least one amino acid from the heavy chain constant region with anamino acid which is different from that present in an unmodified parentantibody, thereby causing an alteration in the binding affinity of theFc domain for a microbial FcPB and/or one or more of the followingproperties: effector function, FcRn binding, serum half-life, stabilityand/or immunogenicity.

The embodiments described herein further provide for a method ofmodifying an antibody of class IgG1 wherein said method includessubstituting at least one amino acid from the heavy chain constantregion selected from amino acid residues (i.e., EU positions) 214, 251,252, 253, 254, 274, 276, 311, 314, 356, 358, 380, 382, 384, 419, 422,428, 431, 432, 433, 434, 435, 436 and 438, thereby causing an alterationin the binding affinity for a microbial FcPB and/or one or more of thefollowing properties: effector function. Additionally, presentdisclosure further provides for a method of producing an antibodyvariant having a heavy chain constant region of mixed isotype, createdby substituting regions of the IgG1 heavy chain constant region withsequences from the IgG3 heavy chain constant region, or a variant IgGincluding a heavy chain constant region of mixed isotype. Suchantibodies and their variants may also contain further modifications, inwhich at least one amino acid from the heavy chain constant region issubstituted with an amino acid residue different from that present inthe IgG1, IgG3 or mixed isotope heavy chain parental antibody. Themethod may include, but is not limited to steps of (a) preparing anexpression vector (e.g., a replicable expression vector) that includes asuitable promoter operably linked to DNA encoding at least a constantregion of an immunoglobulin heavy chain or a variant thereof, wherein atleast one amino acid from the heavy chain constant region is substitutedwith an amino acid which is different from that present in an unmodifiedantibody thereby causing an alteration in FcBP binding affinity and/orone or more of the following properties: effector function, FcRnbinding, serum half-life, stability, and/or immunogenicity antibodies;(b) transforming host cells with said vector; and (c) culturing saidtransformed host cells to produce said modified antibody. Optionally,such a method may further include preparing a second expression vector(e.g., a replicable expression vector) that includes a promoter operablylinked to DNA encoding a complementary immunoglobulin light chain andfurther transforming said cell line with said second vector.

The embodiments described herein also include pharmaceuticalcompositions and methods of prophylaxis and therapy using antibodies andtheir variants, including modified immunoglobulins (includingimmunoglobulins conjugated with antimicrobial compound orradionuclides). Also included are methods of diagnosis using modifiedimmunoglobulins and their variants. In some embodiments, the amino acidmodifications of the present disclosure may be used to enhance theantimicrobial activity of the therapeutic or prophylactic antibody.

Anti-Microbial Immunoglobulins and their Heavy Chain Constant RegionVariants.

According to the embodiments described herein, anti-microbial monoclonalantibodies and their variants are provided. Such anti-microbialmonoclonal antibodies and their variants have variable domains whichrecognize one or more microbial cell surface or secreted antigens.

In some embodiments, IgG antibodies, such as a human IgG antibody, ahumanized or a chimeric IgG class antibody or their variants areclaimed. In such embodiments, the antigen recognition region of theantibody is directed against one or more microbial cell surface orsecreted antigens.

The variant immunoglobulin IgG heavy chain constant region describedherein may be combined with one or more immunoglobulin variable heavyand/or light chain regions which bind antigens produced by microbes thatexpress one or more microbial immunoglobulin binding protein.

In some embodiments, the variable domain of the antibody binds to amicrobial protein that is a microbial immunoglobulin binding protein,and the heavy chain constant region of the antibody is a variant IgGwhich has attenuated binding to one or more microbial Ig Binding Proteinor Ig Binding Protein domain expressed by the target microbe.

In other embodiments, the variable domain of the antibody binds to amicrobial protein that is not an microbial immunoglobulin bindingprotein, and the heavy chain constant region of the antibody is avariant IgG which has attenuated binding to one or more microbial IgBinding Proteins or Ig Binding Protein domains expressed by the targetmicrobe.

The anti-microbial heavy chain constant region variants IgGimmunoglobulins claimed herein have enhanced antimicrobial activityrelative to their parental antibodies.

In some embodiments human, humanized or chimeric anti-microbial heavychain constant region variant immunoglobulins are claimed, whichincludes a heavy chain constant region amino acid sequence selected fromSEQ ID NO: 31-56.

Anti-S. aureus Immunoglobulins and their Heavy Chain Constant RegionVariants.

S. aureus, an important human pathogen for which there is an urgentunmet therapeutic need, a number of microbial immunoglobulin bindingproteins may be expressed, including SpA, Sbi, SSL7 and SSL10.

In some embodiments, the target microbe is S. aureus, and variant IgGantibodies may be designed to have attenuated binding to one or more S.aureus IgBPs due to the introduction of one or more amino acidsubstitutions in the heavy chain constant region relative to theparental IgG.

In other embodiments, the target microbe is S. aureus, and variantantibody heavy chain constant region polypeptide sequences are combinedwith immunoglobulin heavy chain variable polypeptide sequences and lightchains polypeptide sequences, which bind one or more cell surface orsecreted S. aureus antigen.

In some embodiments, the S. aureus antigen recognized by the variabledomain of immunoglobulins and there variants are cell surface orsecreted antigens selected from the list which includes but is notlimited to: ClfA, ClfB, Cna, Eap, Ebh, EbpS, FnBPAK, FnBPB, IsaA, IsaB,IsdA, IsdB, IsdH, SasB, SasC, SasD, SasF, SasG, SasH, SasK, SdrC, SdrD,SdrE, Spa, SraP, Coa, Ecb, Efb, Emp, EsaC, EsxA, EssC, FLIPr, FLIPrlike, Sbi, SCIN-B, SCIN-C, VWbp, SpA, LTA, CP5, CP8, PNAG, dPNAG, CHIPS,PVL leukocidin, α, β and γ-hemolysins, SAK, Sea, Sep, Seb, Epa, Efb,SCIN, Exfoliatins ETB and ETA, Staphylococcal Enterotoxins SEA, SEB,SECn, SED, SEG, SHE, and SEI, Toxic-shock syndrome toxin TSST-1, AlphaToxin, Beta toxin, Delta toxin.

Anti-SpA and Anti-Sbi Immunoglobulins and their Heavy Chain ConstantRegion Variants

In some embodiments, the antigen recognized by the variable domain ofthe antibody or its variants is S. aureus SpA. In such embodiments, themicrobial antigen recognized by the variable domain of the variant IgGantibody is an epitope found in one or more of the repeat homology IgBPdomains of S. aureus SpA (referred to as SpA domains E, D, A, B, and C).

In some embodiments, the antigen recognized by the variable domain ofthe antibody or its variants is S. aureus Sbi. In such embodiments, theantigen epitope recognized by the variable domain of the antibody or itsvariants is located in one or more of the Sbi IgBP binding domains I andII.

In some embodiments, the antigen epitope recognized by the variabledomain of the antibody or its variants is found in two or more of therepeat IgBP homology domains from SpA or Sbi, selected from the list SpAdomains E, D, A, B, and C, and Sbi domains I and II.

In some embodiments, the antigen epitope recognized by the variabledomain of the antibody or its variants is found in one or more of therepeat IgBP homology domains from both SpA and Sbi, selected from thelist SpA domains E, D, A, B, and C, and Sbi domains I and II.

According to the embodiments described herein, anti-SpA monoclonalantibodies and their variants are provided. Such anti-microbialmonoclonal antibodies and their variants, have variable domains whichrecognize S. aureus SpA.

In some embodiments, IgG antibodies, such as a human IgG antibody, ahumanized or a chimeric IgG class antibody and their variants areclaimed. In such embodiments, the antigen recognition region of theantibody is directed against S. aureus SpA.

In one embodiment, methods whereby monoclonal antibodies are raised orselected are provided. The disclosure also envisages the construction ofchimeric antibodies from murine derived antibodies, humanization ofnon-human antibodies and affinity maturation of human or humanizedantibodies. In certain aspects, human and/or humanized SpA antibodiesand variants thereof that are described below may—in addition toaffinity maturation—be subject to one or more maturation mutations thatimprove one or more additional properties in addition to improvingaffinity: avidity, stability, solubility, expression level, and/orbiological activity.

In one embodiment, the murine monoclonal antibody SPA27 (described in WO2008/140487 A2) was used for the construction of chimeric IgGimmunoglobulins and their variants. The heavy chain and light chainvariable domain amino acid sequence of the chimeric antibodies and theirvariants are shown in SEQ ID NO: 1 and 6.

In some embodiment, the murine monoclonal antibody SPA27 and itshumanized versions described in WO 2008/140487 A2 can be used for theconstruction of chimeric IgG immunoglobulins and their variants. Theheavy chain and light chain variable region amino acid sequence of thechimeric antibodies and their variants are shown SEQ ID NO: 1 and 6.

In other embodiments, anti-SpA antibodies known in the art such asmonoclonal antibody 76 (described in U.S. Pat. No. 7,488,807 B2), ormonoclonal antibody 107 (described in US patent application US2010/0047252 A1) can be used for the construction of chimeric, heavychain constant region variant IgG immunoglobulins.

In other embodiments, the CDR sequences of anti-SpA antibodies known inthe art such as monoclonal antibody 3F6, 5A10 and 3D11 (Kim et al.,2012), can be used for the construction of recombinant anti-SpAantibodies (e.g., recombinant murine antibodies), chimeric anti-SpAantibodies, humanized anti-SpA antibodies, or anti-SpA Fc variantantibodies (or anti-SpA heavy chain constant region variant IgGimmunoglobulins) that are derived from a parental antibody (e.g., aparental chimeric anti-SpA antibody, a parental humanized anti-SpAantibody, or a parental human anti-SpA antibody).

According to some embodiments, an anti-SpA antibody includes (i) animmunoglobulin heavy chain, which has a variable heavy chain sequenceand a constant heavy chain sequence; and (ii) an immunoglobulin lightchain, which has a variable light chain sequence and a constant lightchain sequence. In certain aspects, the variable heavy chain includes anamino acid sequence of SEQ ID NO:59, SEQ ID NO:61, or SEQ ID NO:63. Incertain aspects, the variable light chain includes an amino acidsequence of SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:62. Said anti-SpAantibodies may be used to generate a recombinant murine antibody or achimeric antibody. In the case of a chimeric antibody, the variableheavy chain sequence (e.g., SEQ ID NO:59, SEQ ID NO:61, or SEQ ID NO:63)may be combined with a human immunoglobulin (e.g., IgG1) heavy chainconstant sequence to form the chimeric antibody's immunoglobulin heavychain. In some aspects, the chimeric anti-SpA antibody immunoglobulinheavy chain includes an amino acid sequence of SEQ ID NO:71 or SEQ IDNO:72. In addition, the variable light chain sequence (e.g., SEQ IDNO:58, SEQ ID NO:60, or SEQ ID NO:62.) may be combined with a humanimmunoglobulin (e.g., IgG1) light chain constant sequence to form thechimeric antibody's immunoglobulin light chain. In some aspects, thechimeric anti-SpA antibody immunoglobulin light chain includes an aminoacid sequence of SEQ ID NO:65.

In some embodiments, the chimeric antibody described above may be usedas a parental anti-SpA antibody for generating an anti-SpA Fc variantantibody, which includes one or more amino acid substitutions ascompared to the parental chimeric anti-SpA antibody. In such case, theanti-SpA Fc variant antibody may have a variant immunoglobulin heavychain that includes an amino acid sequence of SEQ ID NO:73 or SEQ IDNO:74.

According to other embodiments, a humanized anti-SpA antibody may begenerated. In some aspects the humanized antibody may have a variableheavy chain sequence and a variable light chain sequence which includeone each of a heavy chain CDR1 sequence, a heavy chain CDR2 sequence, aheavy chain CDR3 sequence, a light chain CDR1 sequence, a light chainCDR2 sequence, a light chain CDR3 sequence, each of which may beselected from the CDR sequences in Table 1 below:

Name of Possible Amino Acid Sequence  Sequences Heavy chain GFAFSNYD (SEQ ID NO: 90) CDR1 GFTFNTNA (SEQ ID NO: 91)GYSFTSYY (SEQ ID NO: 92) Heavy chain  ISSGGTYP (SEQ ID NO: 93) CDR2IRSKSNNYAT (SEQ ID NO: 94) IDPFNGGT (SEQ ID NO: 95) Heavy chain (X)GGFLITTRDYYAMDY  CDR3 (SEQ ID NO: 96)* (X)YGYDGTFYAMDY (SEQ ID NO: 97)* (X)EHYDYDYYVMDY  (SEQ ID NO: 98)* Light chain SSVSY (SEQ ID NO: 99) CDR1 ESVEYSGASL (SEQ ID NO: 100) Light chain DTS (SEQ ID NO: 101) CDR2 AAS (SEQ ID NO: 102) EIS (SEQ ID NO: 103)Light chain  QQWSSYPPT (SEQ ID NO: 104) CDR3 QQSRKVPST (SEQ ID NO: 105)QQWSYPFT (SEQ ID NO: 106) *(X) may be substituted with 0, 1, or 2 aminoacids

In some embodiments the (X) of the heavy chain CDR3 sequence may besubstituted with zero (0) amino acids. In other embodiments, the (X) ofthe heavy chain CDR3 sequence may be substituted with two (2) aminoacids, and in some aspects the two amino acids may be selected from theamino acids AR or VT. In some aspects, the humanized anti-SpA antibodymay have an immunoglobulin heavy chain that includes an amino acidsequence of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQID NO:86, or SEQ ID NO:87. In other aspects, the humanized anti-SpAantibody may have an immunoglobulin light chain that includes an aminoacid sequence of SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:85.

In some embodiments, a humanized anti-SpA antibody (such as thosedescribed above), a human anti-SpA antibody or any chimeric anti-SpAantibody may be used as a parental anti-SpA antibody for generating ananti-SpA variant (e.g., a variant heavy chain constant region variant)or an anti-SpA Fc variant antibody. The anti-SpA Fc variant antibody mayinclude an immunoglobulin light chain and a variant immunoglobulin heavychain that has a variable heavy chain sequence and a (variant) constantheavy chain sequence. In such case, the variant immunoglobulin heavychain comprises one or more amino acid substitutions in its constantheavy chain sequence as compared to that of the parental anti-SpAantibody. In some aspects, the anti-SpA Fc variant antibody includes anamino acid sequence selected from SEQ ID NOs: 31-56. In other aspects,the anti-SpA Fc variant antibody includes an amino acid sequenceselected from SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84,SEQ ID NO:88, or SEQ ID NO:89.

In other embodiments, human anti-SpA antibodies can be cloned from humanB cells obtained from patients recovering from a S. aureus infection, orfrom Patients immunized with a non-toxogenic SpA vaccine (Kim et al.,2012).

To compare the effect of variant heavy chain constant region changes onthe effector properties and antimicrobial activity of variant anti-SpAIgG immunoglobulins, control antibodies including parental anti-SpA IgGimmunoglobulins and humanized non-specific antibody having a matchedvariant heavy chain constant region are produced and tested. In oneexample an anti-RSV parental antibody (IgG1, allotype Gm17) having aheavy and light chain sequence shown in HC3 (SEQ ID NO: 22) and LC2 (SEQID NO: 24) or a matched variant heavy chain constant region antibody ofHC4 (SEQ ID NO: 23) and LC2 (SEQ ID NO: 24) were produced and tested.

In some embodiments, chimeric anti-SpA heavy chain constant regionvariant IgG immunoglobulins can be humanized and affinity matured usinga number of established methods, which are known in the art.

In some embodiments, the antigen binding portion of the anti-SpAantibody, or heavy chain constant region variant IgG antibody (i.e., theimmunoglobulin heavy chain of a humanized anti-SpA antibody or variantthereof), contains at least one heavy chain variable region thatincludes an amino acid sequence selected from the group SEQ ID NO: 1-5(VH Chimeric and VH1-VH4).

In some embodiments the antigen binding portion of the antibody, orheavy chain constant region variant IgG antibody (i.e., immunoglobulinlight chain of a humanized anti-SpA antibody or variant thereof),contains at least one light chain variable regions that includes anamino acid sequence selected from the group SEQ ID NO 6-18 (VL chimericand VL1-VL12).

In some embodiments, an anti-SpA antibody or heavy chain constant regionvariant IgG antibody, or antigen-binding portion thereof that includes alight chain variable region amino acid sequence selected form the groupSEQ ID NO: 6-18 (VL chimeric and VL1-VL12), and a heavy chain variableregion amino acid sequence selected from SEQ ID NO:1-5 (VH chimeric andVH1-VH4) are provided.

In one embodiment, a chimeric parental IgG1 anti-SpA antibody thatincludes a heavy chain region acid sequence of SEQ ID NO:19 and a lightchain amino acid sequence of SEQ ID NO:21 is provided.

In one embodiment, an example chimeric variant IgG1 anti-SpA antibodythat includes a variant heavy chain constant acid sequence of SEQ IDNO:20 and a light chain amino acid sequence of SEQ ID NO:21 is provided.

The embodiments described herein also include affinity matured variantanti-S. aureus antibodies in which a human, humanized, or chimericvariable domain of the antibody are derived from an anti-S. aureusantibody. Such claimed affinity matured variant antibodies have at leastone amino acid substitution, deletion or insertion relative to theparental heavy or light chain variable domain sequences.

In some embodiments, the disclosure pertains to an anti-SpA antibody orvariant, or antigen-binding portion thereof that includes a light chainvariable region amino acid sequence selected form the group SEQ IDNO:6-18 (VL chimeric and VL1-VL12), and a heavy chain variable regionamino acid sequence selected from SEQ ID NO:1-5 (VH chimeric andVH1-VH4), in which the antibody variable domain or the heavy and/orlight chain has been affinity matured resulting in the introduction ofvariable region amino acid substitutions, insertions or deletionsrelative to the parental sequence. Such changes result in improvedantibody affinity for its target antigen.

In some embodiments in which the antibody is directed against a S.aureus antigen, the variant immunoglobulins also have low or nosuperantigen type binding to SpA via the Fab region of theimmunoglobulin in addition to one or more heavy chain constant regionchanges that attenuate Fc iterations with one or more S. aureus FcBPs.Such immunoglobulins and their variants can be selected so as to avoidthe use of human VH3 derived sequences, which can interact with SpA at asite distinct from the Fc binding site. Alternatively, if VH3 derivedsequences are used, and Fab-SpA superantigen type binding is present inthe parental immunoglobulin, then modified variable heavy chains areprovided in which at least one amino acid from the heavy chain variableFW region is substituted with an amino acid residue different from thatpresent in the unmodified parental antibody selected from the list of VHresidues including but not limited to H19 and H82a. In some aspects, VHregion variants reduce or abolish the superantigen type binding of theFab region of said variant antibody to S. aureus SpA relative to theparental antibody, but do not significantly attenuate antigen binding tothe antigen binding site of the variant antibody

In an additional embodiment, the modification of human or humanized VH3family derived anti-S. aureus IgG variable heavy domain residues areclaimed which abrogate superantigen type binding of SpA to anti S.aureus immunoglobulins or their heavy chain constant region variants. Inone such embodiment the antigen binding portion of the antibody, orheavy chain constant region variant IgG antibody, contains at least oneheavy chain variable region that includes an amino acid sequenceselected from the group SEQ ID NO:1-5 (VH Chimeric and VH1-VH4), inwhich at least one amino acid from the heavy chain variable region issubstituted with an amino acid residue different from that present inSEQ ID NO:1-5 (VH Chimeric and VH1-VH4), selected from the list of VHresidues (position to Kabat numbering) selected from the list includingH15, H17, H19, H57, H59, H64, H65, H66, H68, H69, H70, H80, H81 and H82(including H82a and other H82 positions).

In an additional embodiment, the modification of human or humanized VH3family derived anti-S. aureus IgG variable heavy domain residues areclaimed which abrogate superantigen type binding of SpA to anti S.aureus immunoglobulins or their heavy chain constant region variants. Inone such embodiment the antigen binding portion of the antibody, orheavy chain constant region variant IgG antibody, contains at least oneheavy chain variable region that includes an amino acid sequenceselected from the group SEQ ID NO:1-5 (VH Chimeric and VH1-VH4), inwhich at least one amino acid from the heavy chain variable FW region issubstituted with an amino acid residue different from that present inSEQ ID NO:1-5 (VH Chimeric and VH1-VH4), selected from the list of VHresidues including but not limited to H19 and H82a (Kabat numbering).

In an additional embodiment, the modification of human or humanized VH3family derived anti-S. aureus IgG variable heavy domain residues areclaimed which abrogate superantigen type binding of SpA to anti S.aureus immunoglobulins or their heavy chain constant region variants. Inone such embodiment the antigen binding portion of the antibody, orheavy chain constant region variant IgG antibody, contains at least oneheavy chain variable region that includes an amino acid sequenceselected from the group SEQ ID NO:1-5 (VH Chimeric and VH1-VH4), inwhich at least one amino acid from the heavy chain variable FW region issubstituted with an amino acid residue different from that present inSEQ ID NO:1-5 (VH Chimeric and VH1-VH4), in which Asn 82a (Kabatnumbering). is either Ser or Gly.

Variant anti-SpA IgG antibodies of the embodiments described herein haveamino acid changes in their heavy chain constant region relative totheir parental antibodies. These amino acid substitutions result in thevariant immunoglobulin having attenuated heavy chain contain domainbinding to one or more microbial immunoglobulin binding protein (IgBP).

In some embodiments, in which the antibody is a variant IgGimmunoglobulin, the microbial antigen recognized by the antibody is S.aureus SpA (SpA), and the antibody is a variant IgG in which amino acidsubstitutions have been introduced into the heavy chain constant regionso as to attenuate Fc binding to one or more S. aureus IgBPs, including,but not limited to: S. aureus SpA, Sbi and SSL10.

In some embodiments, anti-SpA variant antibodies described herein blockone or more function of the SpA domain to which they bind selected from,but not limited to: IgG Fc binding, VH3 Fab binding, TNFR1 binding, vWFbinding, EGFR binding and osteoblast binding.

In some embodiments, the variant Fc domain of the anti-SpA antibody doesnot bind to SpA or Sbi, but will bind to Protein G. Protein G binding ofsuch heavy chain constant region variant anti-microbial immunoglobulinsallows for their purification using Protein G affinity chromatographyusing method well known in the art. In certain embodiments, the variantantibody may bind, via constant domain non-immune binding to Protein Gand/or Protein L, but does not bind SpA or Sbi by interaction with theheavy chain constant domain of the variant antibody.

The disclosure also relates to the prophylactic or therapeutic use ofsuch anti-microbial immunoglobulins and their variants, and their use incombinations with additional antimicrobial chemotherapy oranti-infective agents or in combination with one or more additionalantimicrobial immunoglobulins

The embodiments described herein also include pharmaceuticalcompositions and methods of prophylaxis and therapy using antibodies andtheir variants, including modified immunoglobulins (includingimmunoglobulins conjugated with antimicrobial compound orradionuclides). Also included are methods of diagnosis using modifiedimmunoglobulins and their variants. In some embodiments, the amino acidmodifications of the present disclosure may be used to enhance theantimicrobial activity of the therapeutic or prophylactic antibody

Anti-ClfA Heavy Chain Constant Region Variant Immunoglobulins

In additional embodiments, the antigen recognized by the variable domainof the claimed heavy chain constant region variant immunoglobulin is S.aureus Clumping factor A (ClfA).

In some embodiments a human, humanized or chimeric anti ClfA heavy chainconstant region variant immunoglobulin are claimed.

In some embodiments variant humanized or chimeric anti-ClfA antibodiescontain a heavy chain, in which at least one amino acid from the heavychain constant region selected from, but not limited to amino acidresidues (i.e., EU positions) 214, 251, 252, 253, 254, 274, 276, 311,314, 356, 358, 380, 382, 384, 419, 422, 428, 431, 432, 433, 434, 435,436 and 438 is substituted with an amino acid residue different fromthat present in the unmodified IgG1 antibody.

In some embodiments a human, humanized or chimeric anti ClfA heavy chainconstant region variant immunoglobulin is claimed, including a heavychain constant region amino acid sequence selected from, but not limitedto, SEQ ID NO: 30-56 (heavy chain constant region H1-27).

In one embodiment the heavy and light chain variable domain sequences ofthe humanized anti-ClfA heavy chain constant region variantimmunoglobulin are derived from Tefibazumab.

In one embodiment the heavy and light chain variable domain sequences ofthe humanized anti-ClfA parental and variants including a variable lightchain amino acid sequence SEQ ID NO:29 (VL 13), and a variable heavychain region amino acid sequence SEQ ID NO:28 (VH 5) are provided.

In one embodiments, anti-ClfA heavy chain constant region variantantibodies, including a variable light chain amino acid sequence SEQ IDNO:29 (LC 13), a variable heavy chain region amino acid sequence SEQ IDNO:28 (VH5), and a heavy chain constant region including of an aminoacid sequences selected from the group SEQ ID 30-56 (heavy chainconstant region 1-27) are provided.

In one embodiment, the parental anti-ClfA heavy chain and light chain ofsequence shown in SEQ ID NO:25 (HC 5) and SEQ ID NO:27 (LC 3) areprovided.

In another embodiment, a variant anti-ClfA heavy chain and light chainof sequence SEQ ID NO:26 (HC 6) and SEQ ID NO:27 (LC 3) are provided.

In another embodiment, the heavy and light chain variables domainsequences of the humanized anti-ClfA variant immunoglobulins, haveundergone affinity maturation resulting in at least a 2 fold improvementin its affinity for its antigen.

In another embodiment, an anti-ClfA heavy chain constant region variantIgG immunoglobulin, including a light chain amino acid sequence SEQ IDNO:27 (LC 3), and a variant heavy chain sequence SEQ ID NO:26 (HC6) areprovided. Also claimed are affinity matured derivative immunoglobulinshaving at least one amino acid substitution, deletion or insertionrelative to the parental heavy or light chain variable sequences (SEQ IDNO:29 and SEQ ID NO:28). In one aspect, affinity matured variable domainvariants have an affinity improvement of at lease 2 fold.

In some embodiments, the anti S. aureus activity of the anti-ClfA heavychain constant region variant IgG immunoglobulin and theiraffinity-matured progeny are enhanced relative to their parentalantibodies.

In some embodiments, the variant anti ClfA immunoglobulins describedherein have an increase in one or more of the following Fc mediatedeffector functions: C1q binding, C3b deposition, complement deposition,opsonophagocytic activity, ADCC, ADCP, CDC and anti-microbial activity.

Additional Claimed Embodiments

The embodiments described herein also include heavy chain constantregion variant anti-S. aureus antibodies in which the human, humanized,or chimeric variable domain, or variable domain CDRs of the antibody arederived from an anti-S. aureus antibodies selected from the list:Pagibaximab (a chimeric anti-LTA antibody; Biosynexus/Medimmune),Tefibazumab (a humanized IgG1 anti-ClfA; Aurexis, Inhibitex), CS-D7(human anti-IsdB IgG1, Merck), Aurograb (scFv fragment anti ABCtransporter; NeuTec), anti-Alpha toxin (Medimmune patent applicationWO/2012/109285), mAb15E11, a murine antibody recognizingFibronectin-binding proteins A and B. Povenza et al., 2010).

The embodiments described herein also include affinity matured heavychain constant variant anti-S. aureus antibodies in which the human,humanized, or chimeric variable domain of the antibody are derived fromone or more anti-S. aureus antibodies including, but not limited to:Pagibaximab (a chimeric anti-LTA; Biosynexus/Medimmune; FIG. 37),Tefibazumab (humanized IgG1 anti-ClfA, Inhibitex/BMS), CS-D7 (ahumanized anti-IsdB IgG1, Merck; FIG. 36), Aurograb (an scFv fragmentanti-ABC transporter; NeuTec), and anti-Alpha toxin (Medimmune patentapplication WO/2012/109285, which is hereby incorporated by reference asif fully set forth herein). Such claimed affinity matured heavy chainconstant region variant antibodies have at least one amino acidsubstitution, deletion or insertion relative to the parental heavy orlight chain variable domain sequences.

The antibodies and antibody variants described herein may be of anysuitable antibody structure including, but not limited to, full lengthantibodies, antibody fragments, monoclonal antibodies, bispecificantibodies, multispecific antibodies, peptibodies, intrabodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, fully human antibodies, antibody fusions or Fc fusions(sometimes referred to as “antibody conjugates”), and fragments thereof,respectively. In one embodiment, the antibodies include multispecificantibodies, such as bispecific antibodies, also sometimes referred to as“diabodies”. These are antibodies that bind to two (or more) differentantigens. Diabodies can be manufactured in a variety of ways known inthe art (Holliger & Winter, 1993), e.g., prepared chemically or fromhybrid hybridomas.

Further, the antibodies and antibody variants described herein mayinclude one or more modifications, such as a covalent modification.Covalent modifications of antibodies that are included herein, aregenerally, but not always, done post-translationally. For example,several types of covalent modifications of the antibody are introducedinto the molecule by reacting specific amino acid residues of theantibody with an organic derivatizing agent that is capable of reactingwith selected side chains or the Nor C-terminal residues.

Another type of covalent modification is glycosylation. In anotherembodiment, the IgG variants disclosed herein can be modified to includeone or more engineered glycoforms. An “engineered glycoform,” as usedherein, is a carbohydrate composition that is covalently attached to anIgG, wherein said carbohydrate composition differs chemically from thatof a parent IgG.

Another type of covalent modification of the antibody includes linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337,which are hereby incorporated by reference in their entirety, as iffully set forth herein. In addition, as is known in the art, amino acidsubstitutions may be made in various positions within the antibody tofacilitate the addition of polymers such as PEG. See for example, U.S.Publication No. 2005/0114037, which is incorporated herein by referencein its entirety.

The Fc variants of provided herein are defined according to the aminoacid modifications that compose them. Thus, for example, I332E, orIle332Glu is an Fc variant with the substitution I332E relative to theparent Fc polypeptide. Likewise, S239D/A330L/I332E defines an Fc variantwith the substitutions S239D, A330L, and I332E relative to the parent Fcpolypeptide. It is noted that the order in which substitutions areprovided is arbitrary, that is to say that, for example,S239D/A330L/II332E is the same Fc variant as S239D/1332E/A330L, and soon. For all positions discussed herein, numbering is according to the EUindex or EU numbering scheme (Kabat et al., 1991, Sequences of Proteinsof Immunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, which is hereby incorporated byreference in its entirety as if fully set forth herein). The EU index orEU index as in Kabat or EU numbering scheme refers to the numbering ofthe EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,which is hereby incorporated by reference in its entirety as if fullyset forth herein).

Heavy chain constant region variants may be substantially encoded bygenes from any organism, such as mammals, including but not limited tohumans; rodents including, but not limited to, mice and rats; horses;lagomorpha including, but not limited to, rabbits and hares; camelidaeincluding, but not limited to, camels, llamas, and dromedaries; andnon-human primates including, but not limited to, Prosimians,Platyrrhini (New World monkeys), Cercopithecoidea (Old World monkeys),Hominoidea (including those disclosed in U.S. Patent Publication No.2006/0235208 A1), Gibbons, and Lesser and Great Apes. In one embodiment,the heavy chain constant region variants are substantially human.

The parent heavy chain constant region polypeptide may be an antibody.Parent antibodies may be fully human, obtained for example usingtransgenic mice (Bruggemann et al., 1997) or human antibody librariescoupled with selection methods (Griffiths et al., 1998). The parentantibody need not be naturally occurring. For example, the parentantibody may be an engineered antibody, including but not limited tochimeric antibodies and humanized antibodies (Clark, 2000). The parentantibody may be an engineered variant of an antibody that issubstantially encoded by one or more natural antibody genes. In oneembodiment, the parent antibody has been or can be affinity matured, asis known in the art. Alternatively, the antibody has been modified insome other way, for example as described in U.S. patent application Ser.No. 10/339,788, filed on Mar. 3, 2003, hereby entirely incorporated byreference.

The heavy chain constant region or Fc variants described herein may besubstantially encoded by immunoglobulin genes belonging to any of theantibody classes. In one embodiment, the heavy chain constant regionvariants find use in antibodies or Fc fusions that comprise sequencesbelonging to the IgG class of antibodies, including IgG1, IgG2, IgG3, orIgG4. FIG. 5 provides an alignment of these human IgG sequences. In analternate embodiment the heavy chain constant region variants find usein antibodies or Fc fusions that comprise sequences belonging to the IgA(including subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes ofantibodies. The heavy chain constant region variants described hereinmay comprise more than one protein chain. That is, the presentdisclosure may find use in an antibody or Fc fusion that is a monomer oran oligomer, including a homo- or hetero-oligomer.

It is well known that immunoglobulin polymorphisms exist in the humanpopulation. Gm polymorphism is determined by the IGHG1, IGHG2 and IGHG3genes which have alleles encoding allotypic antigenic determinantsreferred to as Glm, G2m, and G3m allotypes for markers of the humanIgG1, IgG2 and IgG3 molecules (no Gm allotypes have been found on thegamma 4 chain). Markers may be classified into ‘allotypes’ and‘isoallotypes’. These are distinguished on different serological basesdependent upon the strong sequence homologies between isotypes.Allotypes are antigenic determinants specified by allelic forms of theIg genes. Allotypes represent slight differences in the amino acidsequences of heavy or light chains of different individuals. Even asingle amino acid difference can give rise to an allotypic determinant,although in many cases there are several amino acid substitutions thathave occurred. Allotypes are sequence differences between alleles of asubclass whereby the antisera recognize only the allelic differences. Anisoallotype is an allele in one isotype which produces an epitope whichis shared with a nonpolymorphic homologous region of one or more otherisotypes and because of this the antisera will react with both therelevant allotypes and the relevant homologous isotypes (Clark, 1997;Gorman & Clark, 1990).

Allelic forms of human immunoglobulins have been well-characterized (WHOReview of the notation for the allotypic and related markers of humanimmunoglobulins (J Immunogen 1976, 3: 357-362; WHO Review of thenotation for the allotypic and related markers of human immunoglobulins.1976, Eur. J. Immunol. 6, 599-601; Loghem, 1986, all hereby entirelyincorporated by reference). Additionally, other polymorphisms have beencharacterized (Kim et al., 2001). At present, 18 Gm allotypes are known:Glm (1,2,3,17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10,11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bI, c3, b5, bO, b3, b4,s, t, gI, c5, u, v, g5) (Lefranc, et al., The human IgG subclasses:molecular analysis of structure, function and regulation. Pergamon,Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet. 50,199-211, both hereby entirely incorporated by reference). Allotypes thatare inherited in fixed combinations are called Gm haplotypes.

FIG. 7 shows the allotypes of the gamma I chain of human IgG1 and thegamma 3 chain of human IgG3 showing the positions and the relevant aminoacid substitutions (Gorman & Clark, 1990; Jefferis & LeFranc, 2009). Forcomparison, the amino acids found in the equivalent positions in humanIgG2, IgG3 and IgG4 gamma chains are also shown.

The heavy chain constant region or Fc variants described herein may besubstantially encoded by any allotype or isoallotype of anyimmunoglobulin gene. In one embodiment, the heavy chain constant regionvariants may find use in antibodies or Fc fusions that comprise IgG1sequences that are classified as Glm(1), Glm(2), Glm(3), Glm(17),nGlm(I), nGlm(2), and/or nGlm(17). Thus, in the context of an IgG1isotype, the heavy chain constant region variants may comprise a Lys(Glm(17)) or Arg (Glm(3)) at position 214, an Asp356/Leu358 (Glm(1)) orGlu356/Met358 (nGlm(1), and/or a Gly (Glm(2)) or Ala (nGlm(2)) atposition 431 (FIG. 6).

In one embodiment, the heavy chain constant region variants describedherein are based on human IgG1 sequences, and thus human IgG1 sequencesare used as the “base” sequences against which other sequences arecompared including, but not limited to, sequences from other organisms,for example, rodent and primate sequences. Heavy chain constant regionvariants may also comprise sequences from other immunoglobulin isotypes,such as IgG2, IgG3 or IgG4 or from different classes such as IgA, IgE,IgD, IgM, and the like. It is contemplated that, although the heavychain constant region variants of the embodiments described herein areengineered in the context of one parent IgG, the variants may beengineered in or “transferred” to the context of another, second parentIgG. This is done by determining the “equivalent” or “corresponding”residues and substitutions between the first and second IgG, typicallybased on sequence or structural homology between the sequences of thefirst and second IgGs. In order to establish homology, the amino acidsequence of a first IgG outlined herein is directly compared to thesequence of a second IgG. After aligning the sequences, using one ormore suitable homology alignment programs known in the art (e.g., usingconserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first heavy chain constant region variant aredefined. Alignment of conserved residues should conserve 100% of suchresidues. However, alignment of greater than 75% or as little as 50% ofconserved residues is also adequate to define equivalent residues.

Equivalent residues may also be defined by determining structuralhomology between a first and second IgG that is at the level of tertiarystructure for IgGs whose structures have been determined. In this case,equivalent residues are defined as those for which the atomiccoordinates of two or more of the main chain atoms of a particular aminoacid residue of the parent or precursor are within about 0.13 nm andabout 0.1 nm after alignment. Alignment is achieved after the best modelhas been oriented and positioned to give the maximum overlap of atomiccoordinates of non-hydrogen protein atoms of the proteins. Regardless ofhow equivalent or corresponding residues are determined, and regardlessof the identity of the parent IgG in which the IgGs are made, the heavychain constant region variants described herein may be engineered intoany second parent IgG that has significant sequence or structuralhomology with the heavy chain constant region variant. Thus, forexample, if a variant antibody is generated wherein the parent antibodyis human IgG1, by using the methods described above or other methods fordetermining equivalent residues, the variant antibody may be engineeredin another IgG1 parent antibody that binds a different antigen, a humanIgG2 parent antibody, a human IgA parent antibody, a horse IgG7 or IgG4antibody, a mouse IgG2a or IgG2b parent antibody, and the like. Again,as described above, the context of the parent heavy chain constantregion variant does not affect the ability to transfer the heavy chainconstant region variants of the embodiments described herein to otherparent IgGs.

The embodiments described herein provide variant antibodies that areoptimized for a variety of therapeutically relevant properties. A heavychain constant region variant that is engineered or predicted to displayone or more optimized properties is herein referred to as an “optimizedheavy chain constant region variant.” In some embodiments, propertiesthat may be optimized include, but are not limited to, reduced affinityfor one or more microbial IgBP or FcBP. In one embodiment, the variantsof the embodiments described herein may possess similar or enhancedaffinity for a human activating Fcγ R, Fcγ RI, Fcγ RIIa, Fcγ RIIc, FcγRIIIa, and/or FcγRIIIb. In an alternate embodiment, the heavy chainconstant region variants may be optimized to possess reduced affinityfor the human inhibitory receptor FcγRIIb. These embodiments areanticipated to provide IgG polypeptides with enhanced therapeuticproperties in humans—for example, similar or enhanced effector functionrelative to parental IgG and greater anti-microbial potency due toreduced microbial IgBP binding. In other embodiments, Fc of theembodiments described herein may provide enhanced affinity for one ormore FcγRs, and reduced binding to FcRn and microbial IgBPs.

Heavy chain constant region variants of the embodiments described hereinmay comprise modifications that modulate interaction with Fc ligandsother than FcγRs, including but not limited to complement proteins,FcRn, and Fc receptor homologs (FcRHs). FcRHs include but are notlimited to FcRHI, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al.,2002, Immunol. Reviews 190: 123-136, hereby entirely incorporated byreference).

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications may be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an heavy chainconstant region variant of the embodiments described herein. See forexample WO 98/52976; WO 02/079232; WO 00/3317; U.S. Ser. No. 09/903,378;U.S. Ser. No. 10/039,170; U.S. Ser. No. 60/222,697; U.S. Ser. No.10/754,296; PCT WO 01/21823; and PCT WO 02/00165; Mallios, 1999,Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: 942-948;Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO 98/59244; WO02/069232; WO 02/77187; Marshall et al., 1995, J. Immunol. 154:5927-5933; and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358, all ofwhich are hereby entirely incorporated by reference. Sequence-basedinformation can be used to determine a binding score for a givenpeptide-MHC interaction (see for example Mallios, 1999, Bioinformatics15: 432-439; Mallios, 2001, Bioinformatics 17: p942-948; Sturniolo et.al., 1999, Nature Biotech. 17: 555-561, all hereby entirely incorporatedby reference).

In accordance with the embodiments described herein, conventionalmolecular biology, microbiology, and recombinant DNA techniques may beused within the skill of the art. Such techniques are explained fully inthe literature. See, e.g., Sambrook et al, “Molecular Cloning: ALaboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984); all of which are hereby incorporated by reference, asif fully set forth herein.

Methods of Producing Variant Antibodies

The embodiments described herein provide methods for engineering,producing, and screening variant antibodies. The described methods arenot meant to constrain the embodiments described herein to anyparticular application or theory of operation. Rather, the providedmethods are meant to illustrate generally that one or more variantantibodies may be engineered, produced, and screened experimentally toobtain variant antibodies with optimized effector function. A variety ofmethods are described for designing, producing, and testing antibody andprotein variants in U.S. Ser. No. 10/672,280, U.S. Ser. No. 10/822,231,U.S. Ser. No. 11/124,620, and U.S. Ser. No. 11/256,060, all herebyentirely incorporated by reference.

Described herein (see, e.g., Examples 1-4 below) are methods ofproducing monoclonal antibodies that recognize SpA and/or Sbi, methodsfor selecting antibodies that cross react with multiple SpA IgBP domains(selected from Domains E, D, A, B, C and Sbi domains I and II), methodsof selecting antibodies that cross react with one or more SpA IgGbinding domains and/or Sbi domains I and/or II, methods of assaying forantigen binding to SpA or Sbi using variant IgG1 antibodies, having oneor more amino acid substitutions in the Fc domain which prevent Fcbinding to SpA, Sbi or SSL10.

A variety of protein engineering methods may be used to design variantantibodies with optimized effector function. In one embodiment, astructure-based engineering method may be used, wherein availablestructural information is used to guide substitutions. An alignment ofsequences may be used to guide substitutions at the identifiedpositions. Alternatively, random or semi-random mutagenesis methods maybe used to make amino acid modifications at the desired positions.

Methods for production and screening of variant antibodies are wellknown in the art. General methods for antibody molecular biology,expression, purification, and screening are described in AntibodyEngineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg,2001; and Hayhurst & Georgiou, 2001; Maynard & Georgiou, 2000, which arehereby incorporated by reference in their entirety, as if fully setforth herein. Also see the methods described in U.S. Ser. No.10/672,280, U.S. Ser. No. 10/822,231, U.S. Ser. No. 11/124,620, and U.S.Ser. No. 11/256,060, all of which are hereby entirely incorporated byreference.

In one embodiment, the heavy chain constant region variant sequences areused to create nucleic acids that encode the member sequences, and thatmay then be cloned into host cells, expressed and assayed, if desired.These practices are carried out using well-known procedures, and avariety of methods that may find use in the embodiments described hereinare described in Molecular Cloning-A Laboratory Manual, 3rd Ed.(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001),Molecular Cloning-A Laboratory Manual, 4th Ed. (Green and Sambrook, ColdSpring Harbor Laboratory Press, New York, 2012), and Current Protocolsin Molecular Biology (John Wiley & Sons), both entirely incorporated byreference. The variant antibodies of the embodiments described hereinmay be produced by culturing a host cell transformed with nucleic acid,such as an expression vector, containing nucleic acid encoding thevariant antibodies, under the appropriate conditions to induce or causeexpression of the protein. A wide variety of appropriate host cells maybe used, including but not limited to mammalian cells, bacteria, insectcells, and yeast. For example, a variety of cell lines that may find usein the embodiments described herein are described in the ATCC cell linecatalog, available from the American Type Culture Collection. Themethods of introducing exogenous nucleic acid into host cells are wellknown in the art, and will vary with the host cell used.

Purification of Variant Antibodies

In one embodiment, variant antibodies are purified or isolated afterexpression. Antibodies may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, electrophoretic, immunological,precipitation, dialysis, filtration, concentration, and chromatofocusingtechniques. Purification can often be enabled by a particular fusionpartner. For example, proteins may be purified using glutathione resinif a GST fusion is employed, Ni+2 affinity chromatography if a His-tagis employed, or immobilized anti-flag antibody if a flag-tag is used.For general guidance in suitable purification techniques, see AntibodyPurification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag,N Y, 1994, which is hereby entirely incorporated by reference.

In one embodiment, immunoglobulins and their variants are purified byaffinity chromatography on Protein G, Protein L, SpA or by ion exchangechromatography.

Screening Methods

Variant antibodies may be screened using a variety of methods including,but not limited to, those that use in vitro assays, in vivo andcell-based assays, and selection technologies. Automation andhigh-throughput screening technologies may be utilized in the screeningprocedures. Screening may employ the use of a fusion partner or label,for example an immune label, isotopic label, or small molecule labelsuch as a fluorescent or calorimetric dye.

In one embodiment, the functional and/or biophysical properties ofvariant antibodies are screened in an in vitro assay. In anotherembodiment, the protein is screened for functionality, for example itsability to bind to a microbial IgBP or FcBP, its binding affinity to itstarget antigen.

As is known in the art, a subset of screening methods includes thosethat select for favorable members of a library. The methods are hereinreferred to as “selection methods”, and these methods find use in theembodiments described herein for screening variant antibodies. Whenprotein libraries are screened using a selection method, only thosemembers of a library that are favorable, that is which meet someselection criteria, are propagated, isolated, and/or observed. A varietyof selection methods are known in the art that may find use in theembodiments described herein for screening protein libraries. Otherselection methods that may find use in the embodiments described hereininclude methods that do not rely on display, such as in vivo methods. Asubset of selection methods referred to as “directed evolution” methodsare those that include the mating or breading of favorable sequencesduring selection, sometimes with the incorporation of new mutations.

In one embodiment, variant antibodies are screened using one or morecell-based or in vivo assays. For such assays, purified or unpurifiedproteins are typically added exogenously such that cells are exposed toindividual variants or pools of variants belonging to a library. Theseassays are typically, but not always, based on the function of theimmunoglobulin polypeptide; that is, the ability of the immunoglobulinpolypeptide to bind to its target microbial antigen and mediate somebiochemical event, for example effector function, ligand/receptorbinding inhibition, and the like. Such assays often involve monitoringthe response of target cells to the IgG, for example cell killing,change in cellular morphology, opsonophagacytosi, complement deposition,antimicrobial activity. For example, such assays may measure the abilityof variant antibodies immunoglobulins to elicit antimicrobial antigenbinding, microbial killing, and microbial FcBP binding, C1g and C3bdeposition, opsonophagocytosis, ADCC, ADCP, or CDC. For some assaysadditional cells or components, that is in addition to the target cells,may need to be added, for example serum complement, IgG which binds totarget microbial FcBPs, or effector cells such as peripheral bloodmonocytes (PBMCs), NK cells, macrophages, and the like. Such additionalcells may be from any organism, such as humans, mice, rat, rabbit, andmonkey. Antibodies may cause killing of certain microbes, which expressthe target antigen, or they may mediate attack on target microbes byimmune cells, which have been added to the assay. Methods for monitoringtarget cell death or viability are known in the art, and include the useof dyes, immunochemical, cytochemical, and radioactive reagents.

The biological properties of the variant antibodies of the embodimentsdescribed herein may be characterized in cell, tissue, and wholeorganism experiments. As is known in the art, drugs are often tested inanimals, including but not limited to mice, rats, rabbits, dogs, cats,pigs, and monkeys, in order to measure a drug's efficacy for treatmentagainst a disease or disease model, or to measure a drug'spharmacokinetics, toxicity, and other properties. The animals may bereferred to as disease models. Therapeutics are often tested in mice,including but not limited to nude mice, SCID mice, xenograft mice, andtransgenic mice (including knock-ins and knock-outs). Suchexperimentation may provide meaningful data for determination of thepotential of the protein to be used as a therapeutic. Any organism, suchas mammals, may be used for testing. For example because of theirgenetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the IgGs of the embodimentsdescribed herein. Tests in humans may be performed to obtain approval asdrugs. Thus, the IgGs described in the embodiments herein may be testedin humans to determine their therapeutic efficacy, toxicity,immunogenicity, pharmacokinetics, and/or other clinical properties.

In one embodiment, methods of screening and selecting antimicrobialmonoclonal antibodies are provided, the variant heavy chain constantregion used for antibody selection is of human isotype IgG1 having a Histo Arg substitution at position 435 and a Tyr to Phe substitution atposition 436. The variant Fc domain may also be used for antibodyselection is of human isotype IgG1 having a His to Arg substitution atposition 435. The use of such heavy chain constant region variants isimportant, as they allow differentiation between antigen specificbinding by the antibody from heavy chain constant region mediatedbinding to one of the following IgBPs, including but nor limited to SpAand Sbi

In an additional embodiment of screening and selecting antimicrobialmonoclonal antibodies, the variant heavy chain constant region used forantibody selection is of human isotype IgG1 and has a His to Argsubstitution at position 435, a Lys to Gln substitution at position 274and a Tyr to Phe substitution at position 436. In an additional examplethe variant Fc domain used for antibody selection is of human isotypeIgG1 and has a His to Arg substitution at position 435 and a Lys to Glnsubstitution at position 274. The uses of such heavy chain constantregion variants are important so as to differentiate antigen specificvariable domain binding of the antibody from heavy chain constant regionmediated binding to one of the following IgBPs, including but norlimited to SpA, SSL10 and Sbi.

Therapeutic Uses of the Variant Antibodies

The variant antibodies of the embodiments described herein may find usein a wide range of products. In one embodiment an variant antibodydescribed in the embodiments herein is a therapeutic, a diagnostic, or aresearch reagent. The variant may find use in an antibody compositionthat is monoclonal or polyclonal. In one embodiment, variant antibodiesdescribed in the embodiments herein may be used to kill target microbesthat bear the target antigen, for example gram-positive bacterial cells.In an alternate embodiment, the variant antibodies are used to block,antagonize, or agonize the target antigen, for example for antagonizinga bacterial secreted virulence factor. In an alternative embodiment,variant antibodies described herein are used to block or antagonizetarget antigen and kill the target microbe that bear the target antigen.

The anti-microbial variant immunoglobulins described herein, which haveenhanced anti-microbial activity relative to their parental antibodies,may be used for the prophylactic or therapeutic treatment of a number ofimportant infectious diseases infections and pathological conditionscaused by pathogenic microbes. For example, Staphylococcus andStreptococcus bacterial infections are responsible for several diseases,infections, and conditions, such as localized skin infections, diffuseskin infections (e.g., Impetigo), deep, localized infections, acuteinfective endocarditis, septicemia, necrotizing pneumonia, toxinoses(e.g., toxic shock syndrome and staphylococcal food poisoning),cystitis, meningitis, scarlet fever, Rheumatic fever, necrotizingfascitis, and pneumonia. Many of these diseases and conditions are aresult of an opportunistic infection in a patient who has a compromisedimmune system (e.g., from chemotherapy or HIV infection) or an openwound or incision site (e.g., acute injuries or post-surgery)

Therefore, in some embodiments, methods for treating a disease,infection, or condition caused by one or more pathogenic microbesinclude a step of administering a therapeutically effective amount of apharmaceutical composition that includes a variant antibody that hasenhanced antimicrobial effects, such as those described herein. In someaspects, the methods for treating the patient are employed to treat thepatient after the onset of the disease, infection or condition. In otheraspects, the methods for treating the disease are employed to treat thepatient after the onset of the disease, infection or condition as aprophylactic treatment. As such, pharmaceutical composition may includea passive vaccine composition that includes one or more variantantibodies, thereby providing passive immunization to the patient.

A “patient” or “subject” for the purposes of the embodiments describedherein includes humans and other animals, e.g., mammals. The term“treatment” as used herein is meant to include therapeutic treatment, aswell as prophylactic or suppressive measures for a disease, condition ordisorder. Thus, for example, successful administration of apharmaceutical composition that includes a variant antibody of theembodiments described herein prior to onset of the disease results in“treatment” of the disease. As another example, successfuladministration of a pharmaceutical composition that includes a variantantibody of the embodiments described herein after clinicalmanifestation of the disease to combat the symptoms of the disease isconsidered “treatment” of the disease. “Treatment” also encompassesadministration of a pharmaceutical composition that includes a variantof the embodiments described herein after the appearance of the diseasein order to eradicate the disease. Successful administration of apharmaceutical composition that includes a variant of the embodimentsdescribed herein after onset and after clinical symptoms have developed,with possible abatement of clinical symptoms and perhaps amelioration ofthe disease, is considered “treatment” of the disease. Those “in need oftreatment” as used herein, include mammals already having the disease ordisorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

In one embodiment, an variant antibody described herein may beadministered alone (i.e., as the only therapeutically active agent in apharmaceutical composition). In other embodiments, the variant antibodyis administered in combination with one or more additional therapies.The term “in combination” or “in combination with” as used herein, meansin the course of treating at least one disease or condition in a subjectusing two or more therapies (e.g., therapeutic agents, drugs, treatmentregimens, treatment modalities or a combination thereof), in any order.This includes simultaneous administration (or “co-administration”),administration of a first therapy prior to or after administration of asecond therapy, as well as in a temporally spaced order of up to severaldays apart. Such combination treatment may also include more than asingle administration of any one or more therapies. Further, theadministration of the two or more therapies may be by the same ordifferent routes of administration.

Examples of additional therapies that may be administered in combinationwith the variant antibodies described herein include, but are notlimited to, (1) chemotherapeutic agents (e.g., alkylating agents,antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors,mitotic inhibitors hormone therapy, targeted therapeutics andimmunotherapeutics), biological agents, antibodies or variant antibodiessuch as those described herein, antibodies unrelated to those describedherein, antimicrobial agents, antibiotics (e.g., nafcillin, oxacillin,vancomycin, penicillin, ampicillin, aminoglycoside, clarithromycin, orazithromycin), antiviral agents, anti-infective agents, (2) surgery,radiation therapy, or other treatment modalities that may compromise theimmune system, and (3) other suitable therapeutic agents, treatmentmodalities, that may be used to treat a disease, infection or conditioncaused by a pathogenic microbe or an underlying disease or conditionthat is common to patients suffering from a disease, infection orcondition caused by a pathogenic microbe (e.g., cancer patients, surgerypatients, HIV infected patients. In the case where a patient undergoes asurgical procedure or radiation therapy, the variant antibody may beadministered before, during or soon surgery for prophylactic treatmentof opportunistic infections, such as those caused a pathogenic microbe(e.g., Staphyloccus, Streptococcus).

In some embodiments, pharmaceutical compositions are provided wherein anvariant antibody described herein and one or more therapeutically activeagents are formulated as part of a composition. Formulations of thevariant antibodies of the embodiments described herein are prepared forstorage by mixing said IgG having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980,which is hereby incorporated by reference in its entirety, as if fullyset forth herein), in the form of lyophilized formulations or aqueoussolutions. The formulations to be used for in vivo administration arepreferably sterile. This is readily accomplished by filtration throughsterile filtration membranes or other methods. The variant antibody andother therapeutically active agents disclosed herein may also beformulated as immunoliposomes, and/or entrapped in microcapsules.

The concentration of the therapeutically active heavy chain constantregion variant in the formulation may vary from about 0.001 to 100weight %. In one embodiment, the concentration of the IgG is in therange of 0.003 to 1.0 molar. In order to treat a patient, atherapeutically effective dose of the variant antibody of theembodiments described herein may be administered to the patient. A“therapeutically effective dose,” as used herein means a dose thatproduces the effects for which it is administered. The exact dose willdepend on the purpose of the treatment, and will be ascertainable by oneskilled in the art using known techniques. Dosages may range from 0.001to 100 mg/kg of body weight or greater, for example 0.1, I, 10, or 50mg/kg of body weight, with I to 10 mg/kg being a preferred range. As isknown in the art, adjustments for protein degradation, systemic versuslocalized delivery, and rate of new protease synthesis, as well as theage, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

Administration of the pharmaceutical composition that includes a variantantibody of the embodiments described herein, such as those in the formof a sterile aqueous solution, may be done in a variety of ways,including, but not limited to, subcutaneously, intravenously,intranasally, intraotically, transdermally, topically (e.g., gels,salves, lotions, creams, etc.), intraperitoneally, intramuscularly,intrapulmonary (e.g., AERx® inhalable technology commercially availablefrom Aradigm, or Inhance® pulmonary delivery system commerciallyavailable from Inhale Therapeutics), vaginally, parenterally, rectally,or intraocularly. In some embodiments, the pharmaceutical composition isadministered in any of the above routed using a composition in the formof a sterile aqueous solution.

DEFINITIONS

In order that for embodiments be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassequivalents and are not meant to be limiting.

The terms “ADCC” or “antibody dependent cell-mediated cytotoxicity,” asused herein, mean a cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

The terms “ADCP,” or “antibody dependent cell-mediated phagocytosis,” asused herein, mean a cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

The terms “amino acid” and “amino acid identity,” as used herein, meanone of the 20 naturally occurring amino acids or any non-naturalanalogues that may be present at a specific, defined position. The terms“amino acid residue” or “amino acid,” as used herein, refer to aminoacids that are preferably in the “L” isomeric form. However, residues inthe “D” isomeric form can be substituted for any L-amino acid residue,as long as the desired fictional property of immunoglobulin-binding isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxylgroup present at the carboxyl terminus of a polypeptide. In keeping withstandard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

The terms “amino acid modification” or “amino acid substitution” or“substitution,” as used herein, mean an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. An “amino acidsubstitution” or “substitution” as used herein, means a replacement ofan amino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution L328R refers to avariant polypeptide, in this case a heavy chain constant region variant,in which the leucine at position 328 is replaced with arginine. An“amino acid insertion” or “insertion” as used herein means an additionof an amino acid at a particular position in a parent polypeptidesequence. An “amino acid deletion” or “deletion,” as used herein, meansa removal of an amino acid at a particular position in a parentpolypeptide sequence.

Amino acid substitutions can be made by mutation (for example mutationof SEQ ID NO:1-65) such that a particular codon in the DNA sequenceencoding the polypeptide is changed to a codon which codes for adifferent amino acid. Such a mutation is generally made by making thefewest nucleotide changes possible. A substitution mutation of this sortcan be made to change an amino acid in the resulting protein in anon-conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to another grouping) or in aconservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The embodiments described herein should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein.

The following are examples of various groupings of amino acids:

-   -   Amino acids with nonpolar R groups: Alanine, Valine, Leucine,        Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine    -   Amino acids with uncharged polar R groups: Glycine, Serine,        Threonine, Cysteine, Tyrosine, Asparagine, Glutamine    -   Amino acids with charged polar R groups (negatively charged at        Ph 6.0): Aspartic acid, Glutamic acid    -   Basic amino acids (positively charged at pH 6.0): Lysine,        Arginine, Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine.

Another grouping may be according to molecular weight (i.e., size of Rgroups) as shown below:

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

The term “antibody” or “antibodies” as used herein includes full lengthantibodies and antibody fragments, and includes both monoclonal andpolyclonal antibodies. An antibody may also include recombinant,genetically engineered or otherwise modified forms of immunoglobulins,such as intrabodies, peptibodies, minibodies, chimeric antibodies, fullyhuman antibodies, humanized antibodies, bispecific antibodies, andantibody fusions or heteroconjugate antibodies (e.g., diabodies,triabodies, and tetrabodies), An “antibody fragment” as used hereinincludes antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions. Specific antibody fragments mayinclude, but are not limited to, (i) the Fab fragment including VL, VH,CL and CH1 domains, (ii) the Fd fragment including of the VH and CH1domains, (iii) the Fv fragment including of the VL and VH domains of asingle antibody; (iv) the dAb fragment (Ward et al. 1989, Nature341:544-546) which includes of a single variable, (v) isolated CDRregions, (vi) F (ab′) 2 fragments, a bivalent fragment that includes twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., 1988; Huston et al., 1988), (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion(Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; W094/13804;Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448). Theantibody fragments may be modified. For example, the molecules may bestabilized by the incorporation of disulphide bridges linking the VH andVL domains (Reiter et al., 1996).

An antibody typically includes a tetrameric structure. Each tetramer istypically composed of two identical pairs of polypeptide chains, eachpair having one “light” (typically having a molecular weight of about 25kDa) and one “heavy” chain (typically having a molecular weight of about50-70 kDa). Human light chains are classified as kappa and lambda lightchains. Heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. IgG has several subclasses, including, but notlimited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including,but not limited to, IgM1 and IgM2. Thus, “isotype” as used herein ismeant any of the subclasses of immunoglobulins defined by the chemicaland antigenic characteristics of their constant regions. The known humanimmunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1,IgM2, IgD, and IgE.

The terms “CDC” or “complement dependent cytotoxicity,” as used herein,mean a reaction wherein one or more complement protein componentsrecognize bound antibody on a target cell and subsequently cause lysisof the target cell.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cy2”domain) usually extends from about amino acid 231 to about amino acid340. The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain. Burton,Molec. Immunol. 22:161-206 (1985).

The “CH3 domain” includes the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from about amino acid residue 341 to aboutamino acid residue 447 of an IgG).

The terms “chimeric antibody,” “chimeric antibodies,” “humanizedantibody,” and “humanized antibodies” generally refer to antibodies thatcombine antibody regions (scaffold or framework regions and variableregions) from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse (or rat, in somecases) and the constant region(s) from a human. “Humanized antibodies”generally refer to non-human antibodies that have had thevariable-domain framework regions swapped for sequences found in humanantibodies. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:15341536, which are herebyincorporated by reference in their entirety, as if fully set forthherein. “Back mutation” of selected acceptor framework residues to thecorresponding donor residues is often required to regain affinity thatis lost in the initial grafted construct (U.S. Pat. No. 5,530,101; U.S.Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762;U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No.5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). Ahumanized antibody may also comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system (Roué al., 2004). A variety techniques andmethods for humanizing and reshaping non-human antibodies are well knownin the art (See Tsurushita & Vasquez, 2004, Humanization of MonoclonalAntibodies, Molecular Biology of B Cells, 533-545, Elsevier Science(USA), and references cited therein). Humanization methods include butare not limited to methods described in Jones et al., 1986; Riechmann etal., 1988; Verhoeyen et al., 1988; Queen et al., 1989; He et al., 1998;Carter et al., 1992; Presta et al., 1997; Gorman et al., 1991; andO'Connor et al., 1998; which are hereby incorporated by reference intheir entirety, as if fully set forth herein. Humanization or othermethods of reducing the immunogenicity of nonhuman antibody variableregions may include resurfacing methods, as described for example inRoguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969973, which arehereby incorporated by reference in their entirety, as if fully setforth herein. In one embodiment, the parent or variant antibody has beenaffinity matured, as is known in the art. Structure-based methods may beemployed for humanization and affinity maturation, for example asdescribed in U.S. Ser. No. 11/004,590, which is hereby incorporated byreference in its entirety, as if fully set forth herein. Selection basedmethods may be employed to humanize and/or affinity mature antibodyvariable regions, including but not limited to methods described in Wuet al., 1999; Baca et al., 1997; Rosok et al., 1996; Rader et al., 1998and Krauss et al., 2003, which are hereby incorporated by reference intheir entirety, as if fully set forth herein. Other humanization methodsmay involve the grafting of only parts of the CDRs, including but notlimited to methods described in U.S. Ser. No. 09/810,502; Tan et al.,2002, J. Immunol, all of which are hereby entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,502; Tan et al., 2002; De Pascalis et al., 2002,which are hereby incorporated by reference in their entirety, as iffully set forth herein.

The term “constant domain,” as used herein, refers to the portion of animmunoglobulin molecule having a more conserved amino acid sequencerelative to other portions of the immunoglobulin, the variable domain,which contains the antigen binding site. The constant domain containsthe CH1, CH2 and CH3 domain of the heavy chain and the CL domain of thelight chain.

The term “effector function,” as used herein, is a biochemical eventthat results from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include, but are not limited to,Fcγ R-mediated effector functions such as ADCC and ADCP, andcomplement-mediated effector functions such as CDC.

The term “effector cell,” as used herein, is a cell of the immune systemthat expresses one or more Fc receptors and mediates one or moreeffector functions. Effector cells include but are not limited tomonocytes, macrophages, neutrophils, dendritic cells, eosinophils, mastcells, platelets, B cells, large granular lymphocytes, Langerhans'cells, natural killer (NK) cells, and T cells, and may be from anyorganism including but not limited to humans, mice, rats, rabbits, andmonkeys.

The terms “Fab” or “Fab region,” as used herein, mean one or morepolypeptides that comprise the VH, CH1, VL, and CL immunoglobulindomains. Because VL includes the JL region and VH includes the JHregion, JL and JH also compose the Fab region. It is generally viewed inthe art that the Fab region is demarcated N-terminally by the N-terminusand C-terminally by the disulfide bond that covalently links the heavyand light chains. Accordingly, for the purposes of the embodimentsdescribed herein, “Fab region” as used herein includes amino acidspositions from the N-terminus to position 214 of the light chain andfrom the N-terminus to position 220 of the heavy chain, wherein thenumbering of the C-terminal residues is according to the EU numberingscheme. Fab may refer to this region in isolation, or this region in thecontext of a full-length antibody or antibody fragment. Positionaldefinitions of the regions within the Fab, including the VL, VH, JL, JH,CL, and CH1 regions, are illustrated in FIG. 1). The VL kappa and VHregions are well defined genetically and in the art, and accordingly “VLregion” as used herein includes residues 1-107, and “VH region” as usedherein includes residues 1-113, wherein numbering is according to theKabat numbering scheme. The JL kappa region is made up of 5 germ linesequences of equal length, and accordingly, “JL region,” as used herein,includes positions 96-107, wherein numbering is according to Kabat.There are 6 JH germ line sequences of differing length, and the exactKabat position at which this segment combines with the VH germlinevaries. For the purposes of the embodiments described herein, the JHregion may comprise the residues of these sequences that are clearlydefined in a Kabat sequence alignment. Based on this definition, “JHregion” as used herein includes residues 100-113, wherein numbering isaccording to the Kabat numbering scheme. The remaining C-terminal lightand heavy chain sequences of the Fab are made up of the CL and CH1regions respectively. Thus, “CL region” as used herein includespositions 108-214, and “CH1 region” as used herein includes positions118-220, wherein numbering is according to the EU numbering scheme. Fabmay refer to this region in isolation, or this region in the context ofa full-length antibody or antibody fragment.

The terms “Fc Binding protein” or “FcBP,” as used herein, mean amicrobial product that can bind to an immunoglobulin through interactionwith the Fc region of the immunoglobulin. Examples of such proteinsinclude SpA and Protein G which interact with the CH2-CH3 interface ofthe immunoglobulin Fc region, or SSL10 which interacts with IgG1 at sitewhich is distinct from the SpA binding site.

The term “Fc fusion,” as used herein, is a protein wherein one or morepolypeptides are operably linked to Fc. Fc fusion is herein meant to besynonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”,and “receptor globulin” (sometimes with dashes) (Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200, both hereby entirely incorporated by reference). An Fc fusioncombines the Fc region of an immunoglobulin with a fusion partner, whichin general may be any protein, polypeptide or small molecule. The roleof the non-Fc part of an Fc fusion, i.e., the fusion partner, is tomediate target binding, and thus it is functionally analogous to thevariable regions of an antibody. In addition to Fc fusions, “antibodyfusions” include the fusion of the constant region of the heavy chainwith one or more fusion partners or conjugate partners (again includingthe variable region of any antibody), while other antibody fusions aresubstantially or completely full length antibodies with fusion partnersor conjugate partners. In one embodiment, a role of the fusion orconjugate partner is to mediate target binding, and thus it isfunctionally analogous to the variable regions of an antibody (and infact can be). Virtually any protein or small molecule may be linked toFc to generate an Fc fusion or antibody fusion. Protein fusion orconjugate partners may include, but are not limited to, the targetbinding region of a receptor, an adhesion molecule, a ligand, an enzyme,a cytokine, a chemokine, or some other protein or protein domain. Smallmolecule fusion partners may include any therapeutic agent that directsthe Fc fusion to a therapeutic target. Such targets may be any molecule,such as an extracellular receptor that is implicated in disease. Thefusion or conjugate partner can be proteinaceous or non-proteinaceous;the latter generally being generated using functional groups on theantibody and on the conjugate partner. For example, linkers are known inthe art; for example, homo- or hetero-bifunctional linkers as are wellknown (see, 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).

The term “Fc gamma receptor” or “Fcγ R,” as used herein, is any memberof the family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the Fcγ R genes. In humans, this familyincludes, but is not limited to, Fcγ RI (CD64), including isoformsFcγRIa, FcγRIb, and FcγRIc; Fcγ RII (CD32), including isoforms FcγRIIa(including allotypes H131 and R13I), FcγRIIb (including FcγRIIb-1 andFcγRIIb-2), and FcγRIIc; and FcγRIII (CDI6), including isoforms FcγRIIIa(including allotypes VI58 and F158) and FcγRIIIb (including allotypesFcγRIIIb-NAI and Fcγ RIIIb-NA2) (Jefferis et al., 2002, Immunol Lett82:57-65, which is hereby entirely incorporated by reference), as wellas any undiscovered human Fcγ Rs or Fcγ R isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice,rats, rabbits, and monkeys. Mouse Fcγ Rs include but are not limited toFcγ RI (CD64), Fcγ RII (CD32), Fcγ RIII (CDI6), and Fcγ RIII-2 (CDI6-2),as well as any undiscovered mouse Fcγ Rs or Fcγ R isoforms or allotypes.

The term “Fc ligand,” as used herein, is a molecule, such as apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex. Fc ligands include, but arenot limited to, Fcγ Rs, Fcγ Rs, Fcγ Rs, FcRn, Clq, C3, mannan bindinglectin, mannose receptor, staphylococcal SpA, streptococcal protein G,and viral Fcγ R. Fc ligands also include Fc receptor homologs (FcRH),which are a family of Fc receptors that are homologous to the Fcγ Rs(Davis et al., 2002, Immunological Reviews 190:123-136, which is herebyentirely incorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc.

The terms “Fc” or “Fc region,” as used herein, mean a polypeptide thatincludes the heavy chain constant region of an antibody excluding thefirst heavy chain constant region immunoglobulin domain. Thus Fc refersto the last two heavy chain constant region immunoglobulin domains ofIgA, IgD, and IgG, and the last three constant region immunoglobulindomains of IgE and IgM, and the flexible hinge N-terminal to thesedomains. For 1 gA and IgM, an Fc may include the J chain. For IgG, asillustrated in FIG. 1, Fc includes immunoglobulin domains Cgamma2 andCgamma3 (Cγ2 and Cγ3) and the hinge between C gamma 1 (Cγ1) and Cgamma2(Cγ2). Cγ1, Cγ2 and Cγ3 are also commonly referred to as CH1, CH2 andCH3. Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to comprise residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. Fc may refer to this region in isolation, or thisregion in the context of an Fc polypeptide, as described below. The term“Fc polypeptide,” as used herein, is a polypeptide that includes all orpart of an Fc region. Fc polypeptides include, but are not limited to,antibodies, Fc fusions, isolated Fcs, and Fc fragments. Therefore,“outside the Fc region” as used herein means the region of an antibodythat does not comprise the Fc region of the antibody. In accordance withthe aforementioned definition of Fc region, “outside the Fc region” foran IgG1 antibody is herein defined to be from the N-terminus up to andincluding residue T225 or C229, wherein the numbering is according tothe EU numbering scheme. Thus, the Fab region and part of the hingeregion of an antibody are outside the Fc region.

The term “full length antibody,” as used herein, is a structure that isor includes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG isotype is atetramer and includes two identical pairs of two immunoglobulin chains,each pair having one light and one heavy chain, each light chain thatincludes immunoglobulin domains VL and CL; and each heavy chain thatincludes immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals,for example in camels and llamas, IgG antibodies may include only twoheavy chains, each heavy chain including a variable domain attached tothe Fc region.

A “fully human antibody” or “complete human antibody” refers to a humanantibody having the gene sequence of an antibody derived from a humanchromosome with the modifications outlined herein.

The term “germline,” as used herein, is the set of sequences thatcompose the natural genetic repertoire of a protein, and its associatedalleles.

The terms “hinge” or “hinge region,” as used herein, mean the flexiblepolypeptide that includes the amino acids between the first and secondconstant domains of an antibody. Structurally, the IgG CH1 domain endsat EU position 220, and the IgG CH2 domain begins at residue EU position237. Thus for IgG the antibody hinge is herein defined to includepositions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein thenumbering is according to the EU index as in Kabat. In some embodiments,for example in the context of an Fc region, the lower hinge is included,with the “lower hinge” generally referring to positions 226 or 230.

An immunoglobulin Fc variant or heavy chain constant region variantincludes one or more amino acid modifications relative to a parentimmunoglobulin Fc polypeptide or heavy chain constant regionpolypeptide, wherein said amino acid modification(s) provide one or morealtered properties. An Fc or heavy chain constant region variant of theembodiments described herein differ in amino acid sequence from itsparent IgG by virtue of at least one amino acid modification. Thus,variants described herein have at least one amino acid modificationcompared to the parent. Alternatively, the variants described herein mayhave more than one amino acid modification as compared to the parent,for example from about one to fifty amino acid modifications, preferablyfrom about one to ten amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.Thus the sequences of the Fc variants or Ig heavy chain constant regionvariant and those of the parent Fc polypeptide are substantiallyhomologous. For example, the variant heavy chain constant region variantsequences herein will possess about 80% homology with the parent heavychain constant region variant sequence, preferably at least about 90%homology, and preferably at least about 95% homology. Modifications maybe made genetically using molecular biology methods known in the art.

The terms “immunoglobulin BP,” “IgBP” or “microbial immunoglobulinbinding protein,” as used herein, mean a microbial product that can bindto immunologic either through interaction with the Fc region of theimmunoglobulin (e.g. SpA or Protein G), or though non-immune interactionwith the Fab region (e.g. SpA-Fab binding domain), or throughinteraction with heavy or light chain constant regions outside the Fcregion (e.g. L protein of Peptostreptococcus magnus).

The term “immunoglobulin (Ig),” as used herein is a protein includingone or more polypeptides substantially encoded by immunoglobulin genes.

Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full-length antibodies, antibody fragments, and individualimmunoglobulin domains.

The term “IgG,” as used herein, is a polypeptide belonging to the classof antibodies that are substantially encoded by a recognizedimmunoglobulin gamma gene. In humans, this IgG includes the subclassesor isotypes IgG1, IgG2, IgG3, and IgG4. In mice, IgG includes IgG1,IgG2a, IgG2b, IgG3.

The term “isotype,” as used herein, is any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

The term “isotypic modification,” as used herein, is an amino acidmodification that converts one amino acid of one isotype to thecorresponding amino acid in a different, aligned isotype. For example,because IgG1 has a tyrosine and IgG2 a phenylalanine at EU position 296,a F296Y substitution in IgG2 is considered an isotypic modification.

The term “non-immune binding,” as used herein, refers to binding of anantibody to an IgBP virulence factor that does not involveantigen-dependent binding by the variable region of the antibody. Incontrast, the term “immune binding,” as used herein, refers to specificbinding of an antigen by an antibody that involves antigen-dependentbinding by the variable region of the antibody.

The term “novel modification,” as used herein, is an amino acidmodification that is not isotypic. For example, because none of the IgGshave a glutamic acid at position 332, the substitution 1332E in IgG1,IgG2, IgG3, or IgG4 is considered a novel modification.

The terms “parent polypeptide”, “parent protein”, “precursorpolypeptide”, or “precursor protein,” as used herein, mean an unmodifiedpolypeptide that is subsequently modified to generate a variant. Saidparent polypeptide may be a naturally occurring polypeptide, or avariant or engineered version of a naturally occurring polypeptide.Parent polypeptide may refer to the polypeptide itself, compositionsthat comprise the parent polypeptide, or the amino acid sequence thatencodes it. Accordingly, by “parent Fc polypeptide” as used herein ismeant an Fc polypeptide that is modified to generate a variant, and by“parent antibody” as used herein is meant an antibody that is modifiedto generate a variant antibody.

The terms “parental immunoglobulin,” “parental antibody,” or “parentantibody” as used herein, mean an unmodified immunoglobulin polypeptidethat is subsequently modified to generate a variant. Said parentimmunoglobulin polypeptide may be a naturally occurring polypeptide, ora variant or engineered version of a naturally occurring polypeptide.Parental immunoglobulin or antibody may refer to the polypeptide itself,compositions that comprise the parental polypeptide, or the amino acidsequence that encodes it.

The term “position” or “amino acid position,” as used herein, is alocation in the sequence of a protein or an antibody. Positions may benumbered sequentially, or according to established format. Severalformats are known in the art including, but not limited to, EU, Kabat,Chotia, IMGT, AHo, and Abhinandan. One skilled in the art wouldunderstand the corresponding “EU position,” “Kabat position,” “Chotiaposition,” IMGT position,” or “AHo position.” Therefore, any amino acidpositions described herein are for identification purposes only, and arenot meant to be limited to a particular numbering format.

The term “EU position” or “EU numbering” as used herein, is a locationin the sequence of a protein. Positions may be numbered sequentially, oraccording to an established format, for example the EU position(Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969),http://www.imgtorg/IMGTScientificChart/Numbering/IMGTIGVCsuperfamily.html).For example, position 297 is a position in the human antibody IgG1.

The term “Kabat position,” or “Kabat numbering” as used herein, is alocation in the sequence of a protein. Positions may be numberedsequentially, or according to an established format, for example theindex as in Kabat (Kabat et al., “Sequences of Proteins of ImmunologicalInterest”, NIH Publication, 91-3242 (1991),http://www.imgt.org/IMGTScientificChart/Numbering/IMGTIGVCsuperfamily.html).

A “polypeptide” or “protein,” as used herein, is at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides.

The term “residue,” as used herein, is an amino acid, or a position in aprotein and its associated amino acid identity. For example, Asparagine297 (also referred to as Asn297, Asn 297, N297 or 297N) is a residue inthe human antibody IgG1.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and preferably at least about 90 or 95%)of the nucleotides match over the defined length of the DNA sequences.Sequences that are substantially homologous can be identified bycomparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra, which are hereby incorporatedin their entirety as if fully set forth herein.

The term “target cell,” as used herein, is a cell that expresses atarget antigen.

The term “variable region” or “variable domain” as used herein, is theregion of an immunoglobulin that includes one or more Ig domainssubstantially encoded by any of the VK′ VL and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

The terms “variant polypeptide”, “polypeptide variant”, “variantimmunoglobulin”, “variant antibody” or “variant,” as used herein, referto a polypeptide sequence that differs from that of a parentalpolypeptide sequence by virtue of at least one amino acid modificationin any part of an antibody including, but not limited to, the Fc region,the immunoglobulin heavy chain, the heavy chain constant region, theheavy chain variable region, the immunoglobulin light chain, the lightchain constant region, the light chain variable region, or any fragmentof combination thereof. The parent polypeptide may be a naturallyoccurring or wild-type (WT) polypeptide, or may be a modified version ofa WT polypeptide. Variant polypeptide may refer to the polypeptideitself, a composition that includes the polypeptide, or the aminosequence that encodes it. The variant polypeptide may have at least oneamino acid modification compared to the parent polypeptide, e.g. fromabout one to about ten amino acid modifications, or from about one toabout five amino acid modifications compared to the parent. The variantpolypeptide sequence herein may possess at least about 80% homology witha parent polypeptide sequence, at least about 90% homology, or at leastabout 95% homology.

Accordingly, the terms “Fc variant”, or “variant Fc” as used herein,mean an antibody sequence that differs from that of a parent sequence byvirtue of at least one amino acid modification in the Fc region. An Fcvariant may only encompass an Fc region, or may exist in the context ofan antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptidethat is substantially encoded by Fc. Fc variant may refer to the Fcpolypeptide itself, compositions including the Fc variant polypeptide,or the amino acid sequence that encodes it. The terms “Fc polypeptidevariant” or “variant Fc polypeptide,” as used herein, refer to an Fcpolypeptide that differs from a parent Fc polypeptide by virtue of atleast one amino acid modification. The terms “protein variant” or“variant protein,” as used herein, mean a protein that differs from aparent protein by virtue of at least one amino acid modification. Theterms “antibody variant” or “variant antibody,” as used herein, mean anantibody that differs from a parent antibody by virtue of at least oneamino acid modification. The terms “IgG variant” or “variant IgG,” asused herein, mean an antibody that differs from a parent IgG by virtueof at least one amino acid modification. The terms “immunoglobulinvariant” or “variant immunoglobulin,” as used herein, mean animmunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification.

Accordingly, the terms “heavy chain constant region variant”, “variantheavy chain constant region,” “heavy chain constant region variant,” or“variant heavy chain constant region,” as used herein, mean a heavychain constant region antibody sequence that differs from that of aparent sequence by virtue of at least one amino acid modification. Aheavy chain constant region variant may include an heavy chain constantregion alone, or may exist in the context of an antibody, a heavy chainconstant region fusion, isolated heavy chain constant region, heavychain constant region fragment, or other polypeptide that issubstantially encoded by heavy chain constant region. Heavy chainconstant region variant may refer to the heavy chain constant regionpolypeptide itself, compositions that include the heavy chain constantregion variant polypeptide, or the amino acid sequence that encodes it.The terms “heavy chain constant region polypeptide variant” or “variantheavy chain constant region polypeptide,” as used herein, refer to aheavy chain constant region polypeptide that differs from a parent heavychain constant region polypeptide by virtue of at least one amino acidmodification.

Similarly, the terms “heavy chain variable region variant”, “variantheavy chain variable region,” “heavy chain variable region variant,”“variable heavy chain sequence variant,” or “variant heavy chainconstant region,” as used herein, mean a heavy chain variable regionantibody sequence that differs from that of a parent sequence by virtueof at least one amino acid modification. A heavy chain variable regionvariant may include an heavy chain variable region alone, or may existin the context of an antibody, a heavy chain variable region fusion,isolated heavy chain variable region, heavy chain variable regionfragment, or other polypeptide that is substantially encoded by heavychain variable region. Heavy chain variable region variant may refer tothe heavy chain variable region polypeptide itself, compositions thatinclude the heavy chain variable region variant polypeptide, or theamino acid sequence that encodes it. The terms “heavy chain variableregion polypeptide variant” or “variant heavy chain variable regionpolypeptide,” as used herein, refer to a heavy chain variable regionpolypeptide that differs from a parent heavy chain variable regionpolypeptide by virtue of at least one amino acid modification. The terms“protein variant” or “variant protein,” as used herein, mean a proteinthat differs from a parent protein by virtue of at least one amino acidmodification. The terms “antibody variant” or “variant antibody,” asused herein, mean an antibody that differs from a parent antibody byvirtue of at least one amino acid modification. The terms “IgG variant”or “variant IgG,” as used herein, mean an antibody that differs from aparent IgG by virtue of at least one amino acid modification. The terms“immunoglobulin variant” or “variant immunoglobulin,” as used herein,mean an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification.

The term “wild type or WT,” as used herein, is an amino acid sequence ora nucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc. has an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified antibodies. Accordingly, the presentdisclosure provides variant antibodies.

The following examples are intended to illustrate various embodiments ofthe invention. As such, the specific embodiments discussed are not to beconstrued as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope ofinvention, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entirety, as if fully setforth herein.

EXAMPLES

The embodiments described herein is more fully understood by referenceto the following examples. They should not however be construed aslimiting the scope of the invention. All literature and patent citationsmentioned herein are expressly incorporated by reference herein as iffully set forth herein.

The disclosure involves both the generation of anti-microbial variabledomain polypeptides, which constitute the antigen-binding site of theantibody, and their combination with immunoglobulin light and heavychain constant region sequences and their variants. The resultingvariant antibodies have antimicrobial activity. The first section of theexamples covers the generation of variable domain anti-microbialantibodies. In the examples profiles the anti-microbial antibodies aredirected against S. aureus antigens SpA and ClfA. The second sectioncovers the generation of heavy chain constant regions and theirvariants. The third section covers the construction, expression andpurification of antibodies and their variants and the final sectioncovers biological testing of example anti-microbial immunoglobulins andtheir heavy chain constant region variants.

Examples are provided that demonstrate the enhanced anti-microbialactivity of Fc variant anti-Microbial antibodies.

Anti-Microbial Antibody Generation

Anti-S. aureus Antibodies

In some embodiments where the target microbe is S. aureus, heavy chainconstant region variant IgG polypeptide sequences are combined withimmunoglobulin heavy chain variable polypeptide sequences and lightchains polypeptide sequences, which bind one or more cell surface orsecreted S. aureus antigen. Examples of S. aureus antigen recognized bythe variable domain of heavy chain constant region variant IgGantibodies are cell surface or secreted antigens selected from the listwhich includes but is not limited to: ClfA, ClfB, Cna, Eap, Ebh, EbpS,FnBPA, FnBPB, IsaA, IsaB, IsdA, IsdB, IsdH, SasB, SasC, SasD, SasF,SasG, SasH, SasK, SdrC, SdrD, SdrE, Spa, SraP, Coa, Ecb, Efb, Emp, EsaC,EsxA, EssC, FLIPr, FLIPr like, Sbi, SCIN-B, SCIN-C, VWbp, SpA, LTA, CP5,CP8, PNAG, dPNAG, alpha toxin, CHIPS, PVL leukocidin, α, β andγ-hemolysins, SAK, Sea, Sep, Seb, Epa, Efb, SCIN, Exfoliatins ETB andETA, Staphylococcal Enterotoxins SEA, SEB, SECn, SED, SEG, SHE, and SEI,Toxic-shock syndrome toxin TSST-1, Alpha Toxin, Beta toxin, Delta toxin.

As examples of the utility, anti-SpA and anti-ClfA parental and heavychain constant region variant IgG1 antibodies have been generated.Additionally, control heavy chain constant region variant IgG1antibodies which target an unrelated viral antigen (anti-RSV variabledomain) have been produced to enable characterization of microbial IgBPbinding to the heavy chain constant region variants in the absence ofmicrobial binding by the variable domain of the antibody. The followingexamples illustrate the generation of anti-S. aureus antibodies(including humanization of exemplar murine antibodies) and theircombination with example variant heavy chain constant regions describedherein.

Example 1 Epitope Discovery and Generation of Anti-SpA MonoclonalAntibodies (by In Vivo Immunization and Humanization)

In Silico Discovery of Anti-SpA Antigens and Epitopes.

The SpA amino acid sequence from 2 strains of S. aureus (Newman and USA300) was initially examined (FIG. 2). Regions of high inter IgBP domain(SpA domains E, D, A, B and C) sequence homology were found whichprimarily mapped to region of Helices I, II and III. Models of thebinding interfaces of domain B (only Helix I and II are shown forclarity) of SpA with an IgG Fc fragment (FIG. 8, derived from PDB ID: 1FC2) and the SpA domain D with a IgM VH3 Fab fragment (FIG. 9, derivedfrom PDB ID 1 DEE) were constructed from X-ray structures availablewithin the PDB database.

The individual SpA IgBP domains (domains E, D, A, B and C) each adoptthree-helix bundles (FIG. 9 represents the SpA D domain). One face,includes residues from helices I and II binds, IgG Fc (FIG. 8). Residuesfrom helices II and III on the other face bind VH3 Ig (FIG. 9)(Deisenhofer, 1981; Graille et al., 2000).

The amino residues that vary between individual IgBP domains of SpA,referred to as inter-domain variable residues were mapped onto the modeland analyzed (e.g., residues indicated by arrows in FIG. 8 and FIG. 9).

With respect to Fc binding, it was found that inter-domain variableresidues mapped to the face of helix I and II that are not involveddirectly in interactions with the IgG Fc region. Most inter-domainvariable residues are located on the non Fc interacting face of Helix Iand II, the N terminus of Helix I, and the amino acid chain the connectsHelix I and II (FIG. 8).

A similar strategy was taken to analyze residues that are involved ininteraction between SpA IgBP domains and VH3 derived Fab sequences (FIG.3). As shown in FIG. 9, residues involved in the interaction between SpAIgBP Helix II and III of domain D are highly conserved in Domains E, A,B and C. Inter-domain variable residues have been mapped onto the modelshown in FIG. 9 (monomer of SpA domain D and a VH3 Fab fragment). Mostinter-domain variable residues are located on the non-Fab interactingface of Helix II and III.

The amino acid sequence of the individual SpA IgBP domains fromsequenced stains of S. aureus were obtained from public sequence databases and analyzed for intra-domain strain sequence variability. Thealigned sequences are shown in FIGS. 11A-E. The amino acid residuesinvolved with binding to IgFc (FIGS. 8A-B) and VH3 Fab residues (FIGS.9A, B, C) are highly conserved in all sequenced stains (FIGS. 11A-E).This finding demonstrates a high degree of functional (Fc and VH3 Fabbinding residues) conservation of amino acid residues within SpA IgBPdomains of sequenced stains.

In the stains analyzed, no substitutions were found in SpA domain C(FIG. 11C).

Within SpA domains E, D, A and B, inter-strain sequence changes withinindividual SpA domains were highly conservative with respect tofunction. In almost all cases, if a substitution occurred, it is changedto an amino acid that is found in one of the other SpA domains, and theposition is not important for the interacting with either Fc or VH3 Fab(FIGS. 11A-E).

For example a number of stains have a Q to K substitution within HelixIII of SpA domain E (FIGS. 11-E). K is found at the same position in allsequenced stains of S. aureus domains D, A, B and C that were analyzed(FIGS. 11A-D). The position of the Q to K substitution in domain E islocated on the face of Helix III that does not interact with the VH3 Fab(FIG. 10).

Based on the analysis of sequence, a number of different SpA epitopeswere identified that would be highly conserved in SpA domains within aS. aureus strain, and which are also highly conserved between S. aureusstains. These epitopes cover the functionally concerned Fc and Fabbinding faces of SpA domains. Such epitopes, which involve SpA bindinginterfaces required for virulence functions, would be used whentargeting anti-microbial antibodies to S. aureus, as they would be lessprone to the selection of resistance since a) the epitopes are presentin multiple SpA domains, b) mutations within such epitopes are likely toabrogate the virulence function of the SpA domain in which they occur.

Additionally, the repeat nature of the epitope in multiple SpA domainswill enhance antibody avidity. Such antibodies will neutralize one ormore IgBP virulence functions of SpA and target opsono-phagocytosis toSpA coated target microbes.

A number of antibody binding regions of SpA were identified. Theseregions are involved in functional interactions between SpA and Fcand/or Fab. Directing antibody binding to epitopes, which involve thesefunctional binding interfaces, or selecting antibodies with suchproperties, is an important aspect of the embodiments described herein.

One SpA binding region that was identified is the binding interface ofHelix I and II that interacts with IgFc. Epitopes, which involve thisinterface, will be highly conserved between SpA domains and strains.Additionally, monoclonal antibodies recognizing such epitopes will blockFc binding and may also block vWF and TNFR1 binding to SpA domains towhich they bind. In the case in which the epitope to which the antibodybinds involves Helix II, VH3 Fab binding to SpA is also likely to beblocked.

Another SpA binding region that was identified covers Helix II, which isinvolved in interacts with both IgFc and VH3 Fab. Binding of monoclonalantibodies to SpA epitopes within this highly conserved Helix, which canblock both Fc and Fab binding to SpA. As Helix II is virtually invariantbetween SpA domains and between S. aureus stains, this region of SpA isa target of antibodies described herein.

One SpA binding region that was identified is the binding interface ofHelix II and III that interacts with Ig VH3 Fab. Binding of monoclonalantibodies to SpA epitopes within this region are highly conservedbetween SpA domains and strains. Additionally, monoclonal antibodiesrecognizing such epitopes will block VH3 Fab binding and may also blockFc binding to SpA.

Immunization or selection methods for the selection of antibodies thatrecognize conserved SpA epitopes are provided.

The IgFc binding domains of Sbi (I and II) were also analyzed (FIGS. 12,13 and 14). Amino acids within the predicted Helix I region are highlyconserved between Sbi domains I and II (FIG. 12B) and also between Sbidomains and Spa Fc binding helix I and a number of amino acids in HelixII (FIG. 12B). Invariant residues were mapped onto the model of Spadomain D (Helix I and II) binding to the Fc region of an IgG (FIG. 13).As can be seen, residues that interact with IgFc are conserved betweenSpa domains and Sbi domains. In addition to these invariant residues, anumber of highly conservative residues are found in Sbi that are presentin some SpA domains (FIG. 12).

The amino acid sequence of the individual Sbi IgBP domains fromsequenced stains of S. aureus were obtained from public sequencedatabases and analyzed for intra-domain strain sequence variability(FIG. 14). The aligned sequences are shown in FIG. 14 (panel A (Sbidomain I) and panel B (Sbi domain II)). In the stains analyzed, nosubstitutions were found in Sbi domain I (FIG. 14A). Only a singleinter-strain change was found in domain II (FIG. 14B) located withinHelix 1 (N to S substitution in strain CC239_JKD6009). This position isnot conserved between domains I and II, and also differs between SpAdomains E and the residue found in domains D, A, B and C. Mapping of thelocation of this residue onto the SpA model shows that this residue isnot directly involved in interaction with the IgG Fc binding interface(FIG. 15).

Thus, the Fc binding interface of Sbi and SpA are conserved within andbetween S. aureus strains. The conserved Fc binding interfaces of Sbiand SpA domains are attractive targets for raising monoclonalantibodies. Such antibodies will neutralize one or more virulencefunctions of the SpA or Sbi domain to which they bind. Additionally,such antibodies will have anti S. aureus activity.

Example 2 Murine, Human or Humanized Antibodies Generation

Immunization or selection methods for the selection of cross-reactiveantibodies (i.e. antibodies that recognize multiple SpA IgBP domainsand/or Sbi domains) are provided.

In the case where antibodies are derived from wild type mice, such asfemale Swiss Webster Mice, the murine monoclonal antibodies selected maybe humanized by CDR grafting using methods known in the art (Almagro andFransson (2008)).

In an alternative method, human antibodies can be obtained directlyusing transgenic mice methods such as VelocImmune®. VelocImmune® is amouse with a genetically humanized immune system, which can be used togreatly increase the speed and efficiency of in-vivo generation offully-human therapeutic antibodies (Lonberg N (2005)).

In yet an alternative method, antibody domain fragments can be selectedusing Display technologies such as phage, yeast and ribosome displayusing methods known in the art. Following reformatting, such antibodiescan be affinity matured, if required, to high affinity by methods knownin the art (Hoogenboom HR (2005)).

Alternatively, human antibody variable domains can be selected frommammalian display libraries using methods known in the art. Suchantibodies can be affinity matured to high affinity by methods known inthe art (Bowers et al., 2011).

An alternative antibody discovery method exploits high-throughput DNAsequencing to analyze the VL and VH gene repertoires derived from themRNA transcripts of fully differentiated mature B cells orantibody-secreting Bone Marrow Plasma Cells from SpA immunized mice asdescribed by Reddy (Reddy et al, 2010). After a bioinformatics analysis,abundant VL and VH gene sequences are identified within the repertoireof each immunized mouse. VL and VH genes are then paired according totheir relative frequencies within the repertoire. Antibody VH and VLgenes are synthesized by oligonucleotide and PCR assembly. Recombinantantibodies can be expressed in bacterial and mammalian systems assingle-chain variable fragments (scFv), or full-length chimeric variantIgG1 antibodies respectively. Antibodies of interest can then behumanized and affinity matured using methods know to those practicingthe art (CDR grafting followed by affinity maturation).

In an alternative method, human antibodies can be isolated from patientsor volunteers following recovery from a microbial infection orimmunization with a vaccine against the target microbe (Wrammert et al.,2008)

Example 3 In Vivo Immunization and Selection of Anti-SpA and Anti-SbiAntibodies

Immunization Protocol: Mice may be used in this procedure. The SpAantigen used as an antigen for immunization can be obtained from anumber of commercial sources or by standard molecular biology methodsknown in the art. Alternatively, recombinant SpA (e.g. Thermo FisherScientific cat #21184) or individual Ig binding domains or combinationsof SpA domains (selected from the list: SpA domain A, B, C, D or E), canbe produced using standard recombinant technology. Immunization of wildtype or transgenic animals (Lonberg N (2005), Almagro and Fransson(2008)) are effective method for generating antibodies to many antigens.

For antibody screening following immunization, subjects can be bled twoweeks after each immunization booster and non-pooled samples can bechecked for anti-SpA antibodies according to the protocol describedbelow. Due to the interaction of murine and human isotypes with SpA viathe Fc domain, murine IgG1 or human IgG3 are often used for screening toavoid interference from non-immune IgBP binding to the SpA antigen.

The ELISA format that can be used to screen for antibodies is asfollows: In one example ELISA plates (e.g. Nunc MaxiSorp 96 well plates)are coated with goat anti murine IgG1 antibody and then blocked usingthe manufacturers recommended method. Following washing, dilutions ofmurine serum samples are added to wells and incubated. Following washingof the plates to remove unbound materials, peroxidase conjugatedantigen, such as SpA or Sbi, is added to plates. In the case of anti-SpAmurine antibodies, Mild Elution Buffer pH 6.0 (Thermo Scientific cat#21033) is used for plate washing. At this pH, binding of SpA to murineIgG1 via the Fc domain of the antibody minimal. Following washing at pH6.0 with Mild Elution Buffer pH 6.0 (Thermo Scientific cat #21033) toremove unbound conjugate, anti-SpA murine IgG1 is detected usingstandard Peroxidase reagents and the absorbency signal is read.

Alternatively, engineered SpA antigens can be used which contains pointmutations (Kim et al., 2010, 2102) which abolishes Fc binding to domainsA, B, C, D and E of SpA (SpA KK containing the following substitutionsin each SpA domain: Q9K and Q10K or SpA KKAA containing the followingsubstitutions in each SpA domain: Q9K, Q10K, D36A and D37A (Kim et al.,2010)). To determine SpA specific serum IgG, affinity purified SpA KK orSpA KKAA can be used to coat ELISA plates (NUNC Maxisorp) at 1 μg·ml−1in 0.1 M carbonate buffer (pH 9.5 at 4° C.) overnight. The followingday, plates are blocked and incubated with dilutions of hyperimmune seraand developed using OptEIA reagent (BD Biosciences).

Fusion Protocol.

The mouse selected for fusion is boosted with the same dose of antigenused in previous immunizations. The booster regime may be administeredover the four-day period prior to splenectomy and cell fusion.Alternatively, the animal can be boosted with recombinant proteinconsisting of individual IgBP SpA domains from the same of a differentS. aureus strain, or combinations of domains selected from the list: SpAdomain A, B, C, D or E.

In another strategy designed to identify antibodies that cross-reactwith multiple SpA domains, the primary immunization uses one isolatedSpA domain, and the booster includes a different domain or domains thanused for the primary immunization. The booster regime may beadministered over the four-day period prior to splenectomy and cellfusion.

In another strategy designed to identify antibodies that cross reactwith SpA and Sbi, the booster can be recombinant IgBP Sbi domain I, IIor I and II. Such a strategy is designed to select for antibodies thatrecognize a conserved interaction interface between both SpA and Sbi andFcγ.

In yet another immunization strategy, Domain I and II of Sbi can be usedfor the primary immunization, and SpA, or its individual Ig bindingdomains selected from the list domain A, B, C, D or E, can be used as abooster. Such a strategy will select for antibodies that cross reactingwith epitopes that are found on the FcBP domains of Sbi domain I or IIand one or more Spa domains selected from the list: domain A,B,C,D andE. Such a strategy is designed to select for antibodies that recognize aconserved interaction interface between both SpA and Sbi and Fcγ.

On the day of fusion the selected mouse is sacrificed and the spleen isremoved aseptically. The spleen may be minced using forceps and strainedthrough a sieve. The cells may be washed twice using IMDM medium(Iscove's Modified DMEM with L-glutamine and 25 mM HEPES, Cellgrocatalog number 10-016-CM; Mediatech, Inc., Herndon, Va.) and countedusing a hemocytometer. The mouse myeloma cell line should be removedfrom static log-phase culture. The cell are washed with IMDM twice andcounted using a hemocytometer.

Myeloma and spleen cells should then be mixed in a 1:5 ratio andcentrifuged. The supernatant is discarded. The cell pellet is thengently resuspended by tapping the bottom of the tube. One milliliter ofa 50% solution of PEG (MW 1500) is added (drop by drop) over a period of30 seconds. The pellet is mixed gently for 30 seconds using a pipette.The resulting cell suspension is allowed to stand undisturbed foranother 30 seconds. One milliliter (mL) of IMDM is then added over aperiod of one minute, followed by the drop wise addition of two mL ofIMDM over a period of two minutes. Another five mL of IMDM is addedimmediately the two-minute period. The resulting cell suspension may beleft undisturbed for 5 minutes.

The cell suspension may be centrifuged at room temperature for 10minutes at 1200 rpm. The pellet is then resuspended in HAT medium (IMDMcontaining 10% FBS, 2 mM L-glutamine, 0.6% 2-mercaptoethanol (0.04%solution), hypoxanthine, aminopterin, thymidine, and 10% Origen growthfactor). The cells are resuspended to 1×10⁶ cells per milliliter. Cellsuspensions are plated into 96-well plates. Two hundred microliters (orapproximately 2×10⁵ cells) are added to each well. The 96-well platesare incubated at 37° C. in a 7% CO₂ atmosphere with 100% humidity.

Seven days after the fusion, the media should be removed and replacedwith IMDM containing 10% FBS, 2 mM L-glutamine, 0.6% 2-mercaptoethanolstock (0.04%), hypoxanthine and thymidine.

Hybridoma Expansion Protocol.

Fourteen days after fusion, the supernatant may be taken from wells withgrowing hybridoma colonies. The volume of supernatant in each well maybe approximately 150-200 microliters. This supernatant may be tested forIgG1 isotype producing hybridomas with specificity for SpA using ELISAas described herein.

Positive hybridoma colonies may be transferred from the 96-well plate toa 24-well plate and 1.8 mL of IMDM containing 20% FBS, 10% OrigenCloning Factor, 2 mM L-glutamine and 0.6% 2-mercaptoethanol stock(0.04%) is added to each well. The 24-well plates are incubated asdescribed for the 96-well plates above. Five days later, the supernatantfrom 24-well plate should be tested to confirm the presence of specificantibody.

Cells from positive wells may be expanded in T-25 and T-75 flasks(Corning Flasks, Corning, N.Y.). Five vials (1 mL each) of the cellsfrom T-75 flasks are frozen in liquid nitrogen. Cells from positivewells can be cloned by limiting dilution, i.e., hybridoma cells areplated onto 96-well plates at a density of 0.25 cells per well. Growingcolonies may be tested 10-14 days later using the same assay that wasused to initially select the hybridomas. The subcloned cells areexpanded to 24-well plates and, subsequently, T-25, T-75 and T-162flasks. Vials of subclone cells are frozen as described above.

Sequencing of monoclonal antibodies: Total RNA samples from hybridomacells were isolated using a standardized protocol. Briefly, 1.4×10⁷hybridoma cells cultured in DMEM-10 medium with 10% fetal bovine serum(FBS) were washed with PBS, sedimented by centrifugation, and lysed inTRIzol (Invitrogen). Samples were mixed with 20% chloroform andincubated at room temperature for 3 min and centrifuged at 10,000×g for15 min at 4° C. RNAs in the aqueous layer were removed and washed with70% isopropanol. RNA was sedimented by centrifugation and washed with75% diethylpyrocarbonate (DEPC)-ethanol. Pellets were dried and RNAdissolved in DEPC. cDNA was synthesized with the cDNA synthesis kit(Novagen) and PCR amplified using the PCR reagent system (Stratagene),independent primers (5 pmol each), and a mouse variable heavy and lightchain-specific primer set (Novagen). PCR products were sequenced andanalyzed using IMGT/V-QUEST(http://www.imgt.org/IMGT_vquest/share/textes/).

Example 4 Generation of Chimeric and Humanized Anti-Microbial IgG1Antibodies

Anti SpA Antibodies:

In one example, a chimeric parental version of the murine SPA27 antibodywas constructed using the murine variable domain sequences as publishedin patent application WO 208/140487 A2. The murine variable heavy chain(VH chimeric (SEQ ID NO: 1)) was combined with a human IgG1 heavy chainconstant sequence of allotype G1m17 (SEQ ID 30). IgG1 allotype G1m17,1,2has been used as the reference. The allotypic amino acid positions thatinclude a residue substitution relative to the reference sequence areshown Bold underlined in SEQ ID 30. The heavy chain amino acid sequenceof the resulting chimeric antibody is shown in HC 1 (SEQ ID NO: 19).Likewise, the murine variable light chain sequence (VL chimeric (SEQ IDNO: 6)) was combined with a Kappa light chain constant region ofallotype Km3, resulting in a chimeric light chain amino acid sequence asshown in LC 1 (SEQ ID NO:21). Heavy chain constant region variantantibodies were constructed as described above for parental antibodies.Following codon optimization for mammalian expression, DNA encoding aKozak sequence, an N-terminal leader secretion sequence and the targetpolypeptides was synthesized, cloned into a mammalian expression vectorpTT5, and expressed in HEK 293 cells using methods well known in the art(described later).

CDR Grafting and Humanization of Chimeric Antibodies:

CDR grafting can be used to humanize murine antibodies using standardmolecular biology techniques known in the art. In one example CDRgrafting was used to humanize anti-SpA murine antibody sequences usingstandard molecular biology techniques known in the art. Such graftedantibodies sequences (humanized) will generally require additionalaffinity maturation to arrive at a therapeutic humanized antibody ofsufficient affinity. Standard methodology known to those practicing theart can be used for both CRD grafting and affinity maturation. Oneexample is the mouse HC and LC CDR sequences from DNA encoding theanti-SpA monoclonal antibody SPA27.

CDRs were grafted into a human IgG1 heavy chain and a Kappa lightantibody backbone sequences. The selection of the variable domain humangerm line sequence used for grafting is determined by the closesthomology to the mouse hybridoma variable domain sequence. In some cases,different VH germ line sequences can be used for each FW region. In thecase of SPA-27, the closest heavy chain matches are VH3-49 and VH3-72.Variable heavy chain sequences were combined with constant heavy chainsequences from human IgG1 or its variants. Heavy chain constant regionvariants which do not bind SpA are used for screening chimeric, CDRgrafted and affinity mutated antibodies so as to avoid SpA-Fc binding inELISA assays, and to allow binding measurements using ELISA, BIACore orDLS (Dynamic Light Scattering).

Design and Construction of Humanized Antibodies Using the Murine SPA27Anti-SpA Antibody Variable Region Sequence:

Using the anti-SPA27 murine monoclonal antibody as a reference, anti-SPAantibodies were designed using CDR grafting technology. The grafted CDRregions of the variable domains were then combined with light and heavychain variant human IgG1 constant regions sequences.

The sequences of the heavy and light chain variable regions of SPA-27were compared to human germline databases and homologous sequences wereidentified. CDR grafted human Antibody sequences (SEQ ID #1-16) wereinitially designed. CDR grafted antibodies comprise target variableregions derived from either VH3-49, VH3-72 or VH3-70 human germ lineantibody sequences. In an alternative approach, CDR grafted antibodiescan comprise a mixture of sequences derived from VH3-49, VH3-72 orVH3-70. A summary of the CDR grafted Human Antibodies derived fromSPA-27 are given below.

Heavy Chain Variable Domain.

A Summary of the CDR grafted antibodies sequences are given below. Thesequence for each variable heavy chain region is given:

VH chimeric (SEQ ID NO: 1)EVKLVESGGGLVQPGGSRRLSCTTSGFTFTESFMTWVRQPPGKALDWLAFIRNKANGYTTEYSASVKGRFTIARDNSQSILYLQMNALRAEDSATYYCVRGGEYPLYVMDYWGKGTSVTVSS VH1 (SEQ ID NO: 2):EVQLVESGGGLVQPGRSLRLSCTASGFTFTESFMSWFRQAPGKGLEWVGFIRNKANGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCVRGGEYPLYVMDYWGQGTLVTVSS VH2 (SEQ ID NO: 3):EVQLVESGGGLVQPGRSLRLSCTASGFTFTESFMSWIRQPPGKALEWLAFIRNKANGYTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCVRGGEYPLYVMDYWGQGTLVTVSS VH3 (SEQ ID NO: 4):EVQLVESGGGLVQPGGSLRLSCAASGFTFTESFMDWVRQAPGKGLEWVGRIRNKANGYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRGGEYPLYVMDYWGQGTLVTVSS VH4 (SEQ ID NO: 5):EVQLVESGGGLVQPGGSLRLSCAASGFTFTESFMDWIRQPPGKALEWLAFIRNKANGYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRGGEYPLYVMDYWGQGTLVTVSS

Light Chains.

A Summary of the CDR grafted antibodies is given below. The sequence foreach variable light chain region is given:

VL chimeric (SEQ ID NO: 6):DIVLTQSPVSLAVSLGQRATISCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDFATYFCQQSRKVPW TFGGGTKLEIKVL1 (SEQ ID NO: 7): DIVMTQSPDSLAVSLGERATINCKSSESVEYYDTSLLAWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKVPW TFGQGTKLEIKVL2 (SEQ ID NO: 8): DIVMTQSPDSLAVSLGERATINCKSSESVEYYDTSLLAWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFTLTISSLQEEDVAVYYCQQSRKVPW TFGQGTKLEIKVL3 (SEQ ID NO: 9): DIVMTQSPDSLAVSLGERATINCKSSESVEYYDTSLLAWYQQKPGQPPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDVAVYYCQQSRKVPW TFGQGTKLEIKVL4 (SEQ ID NO: 10): DIVMTQSPDSLAVSLGERATINCKSSESVEYYDTSLLAWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFTLTISSLQPEDVAVYYCQQSRKVPW TFGQGTKLEIKVL5 (SEQ ID NO: 11): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKVPW TFGQGTKLEIKVL6 (SEQ ID NO: 12): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFTLTISSLQEEDVAVYYCQQSRKVPW TFGQGTKLEIKVL7 (SEQ ID NO: 13): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQEEDVAVYYCQQSRKVPW TFGQGTKLEIKVL8 (SEQ ID NO: 14): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFTLTISSLQPEDVAVYYCQQSRKVPW TFGQGTKLEIKVL9 (SEQ ID NO: 15): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQEEDFATYFCQQSRKVPW TFGQGTKLEIKVL10 (SEQ ID NO: 16): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFTLTISSLQEEDFATYFCQQSRKVPW TFGQGTKLEIKVL11 (SEQ ID NO: 17): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQEEDFATYFCQQSRKVPW TFGQGTKLEIKVL12 (SEQ ID NO: 18): DIVMTQSPDSLAVSLGERATINCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFTLTISSLQPEDFATYFCQQSRKVPW TFGQGTKLEIK

The humanized variable heavy chain sequences (SEQ ID NO:2-5)) werecombined with a human IgG1 heavy chain constant sequence of allotypeG1m17 (SEQ ID NO:30) to generate a humanized heavy chain sequence.Likewise, the humanized light chain sequences (SEQ ID NO:7-18) werecombined with a Kappa light chain constant region of allotype Km3,resulting in humanized light chain amino acid sequences. Heavy chainconstant region variant antibodies were designed and constructed asdescribed above for parental antibodies. Following codon optimizationfor mammalian expression, DNA encoding a Kozak sequence, an N-terminalleader secretion sequence and the target polypeptides was synthesized,cloned into a mammalian expression vector pTT5, and expressed in HEK 293cells using methods well known in the art (described later).

Anti SpA Example Antibodies:

An anti SpA chimeric parental antibody was constructed as follows. Themurine variable heavy chain (VH chimeric (SEQ ID NO:1)) was combinedwith a human IgG1 heavy chain constant sequence of allotype G1m17 (SEQID NO:30). The heavy chain amino acid sequence of the resulting chimericantibody is shown in HC 1 (SEQ ID NO:19). Likewise, the murine variablelight chain sequence (VL chimeric (SEQ ID NO:6)) was combined with aKappa light chain constant region of allotype Km3, resulting in achimeric light chain amino acid sequence as shown in LC 1 (SEQ IDNO:21). Heavy chain constant region variant antibodies were constructedas described above for parental antibodies. In one example a heavy chainconstant region variant (SEQ ID NO:40) constructed. Antibody and theirvariants were expressed as essentially as follows: Following codonoptimization for mammalian expression, DNA encoding a Kozak sequence, anN-terminal leader secretion sequence and the target polypeptides wassynthesized, cloned into a mammalian expression vector pTT5, andexpressed in HEK 293 cells using methods well known in the art(described later).

Anti-ClfA Antibodies:

A humanized version of the anti-ClfA antibody T1-2 (Domanski et al.,2005) was constructed using the variable domain sequences as publishedin patent application U.S. Pat. No. 6,979,446B2. A parental controlantibody sequence was generated as follows: The variable heavy chainfrom antibody T1-2 (SEQ ID NO:28) was combined with a human IgG1 heavychain constant sequence of allotype G1m17(SEQ ID NO:30). The heavy chainamino acid sequence of the resulting antibody is shown in HC 5 (SEQ IDNO: 25. Likewise, the variable light chain sequence (VL chimeric (SEQ IDNO: 29)) was combined with the a Kappa light chain constant region ofallotype Km3, resulting in a chimeric light chain amino acid sequence asshown in LC 2 (SEQ ID NO:24). Heavy chain constant region variantantibodies were designed and constructed as described above for parentalantibodies. In one example a heavy chain constant region variant (SEQ IDNO:40) constructed. Antibody and their variants were expressed asessentially as follows: Following codon optimization for mammalianexpression, DNA encoding a Kozak sequence, an N-terminal leadersecretion sequence and the target polypeptides was synthesized, clonedinto a mammalian expression vector pTT5, and expressed in HEK 293 cellsusing methods well known in the art (described later).

Anti-RSV Control Antibodies:

A parental humanized anti-RSV antibody derived from Palivizumab(Synagis, Medimmune Inc) has been used as a heavy chain constant regioncontrol antibody (parental polypeptide sequence is shown in SEQ 22). Theanti-RSV parental antibody and its heavy chain constant region variantsallow the effects of variants to be studies in the absence of targetmicrobe binding by the antigen binding variable domain. The parentalhumanized anti-RSV heavy chain variable domain was combined with a humanIgG1 heavy chain constant region of allotype G1m17 resulting in an aminoacid sequence HC3 (SEQ ID NO:22). Likewise, the anti-RSV variable lightchain was combined with a Kappa light chain constant region of allotypeKm3, resulting in a chimeric light chain amino acid sequence of LC 2(SEQ ID NO:24). Heavy chain constant region variant antibodies weredesigned and constructed as described above for parental antibodies. Inone example a heavy chain constant region variant (SEQ ID NO:40)constructed. Antibody and their variants were expressed as essentiallyas follows: Following codon optimization for mammalian expression, DNAencoding a Kozak sequence, an N-terminal leader secretion sequence andthe target polypeptides was synthesized, cloned into a mammalianexpression vector pTT5, and expressed in HEK 293 cells using methodswell known in the art (described later).

Generation of Heavy Chain Constant Region Antibodies and their VariantsExample 5 Anti-Microbial Heavy Chain Constant Region Variants

Parental and heavy chain constant region variant anti-SpA, anti-Clfa andanti-RSV antibodies were constructed as described above for parentalantibodies. In such variant antibodies, the heavy chain constant regionof the variant antibody, contains a heavy chain constant regionsincluding an amino acid sequences selected from the group SEQ IDNO:30-56 (Heavy chain constant region 1-27).

In one example a parental chimeric anti-SpA antibody and an exampleheavy chain constant region variant (SEQ ID NO: 40) antibody wereexpressed, purified and characterized: Shown are the amino acid sequenceof an anti-SpA parental heavy chain (SEQ ID NO: 19), a variant heavychain (SEQ ID NO: 20) and a common light chain (SEQ ID NO: 21). Aminoacid differences between the parental antibody of allotype G1m17 and anexample variant heavy chain content chain amino acid sequence are shownin Bold underlined:

Anti SpA Chimeric HC G1M17 HC 1 (SEQ ID NO: 19)EVKLVESGGGLVQPGGSRRLSCTTSGFTFTESFMTWVRQPPGKALDWLAFIRNKANGYTTEYSASVKGRFTIARDNSQSILYLQMNALRAEDSATYYCVRGGEYPLYVMDYWGKGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKAnti SpA Chimeric variant HC G1M17 HC 2 (SEQ ID NO: 20)EVKLVESGGGLVQPGGSRRLSCTTSGFTFTESFMTWVRQPPGKALDWLAFIRNKANGYTTEYSASVKGRFTIARDNSQSILYLQMNALRAEDSATYYCVRGGEYPLYVMDYWGKGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLSP GKAnti SpA Chimeric LC LC 1 (SEQ ID NO: 21)KM3DIVLTQSPVSLAVSLGQRATISCRASESVEYYDTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDFATYFCQQSRKVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC

As control antibodies, a humanized RSV variable domain has been used,derived from Palivizumab (Synagis, Medimmune Inc). This anti-RSVvariable domain allows the effects of heavy chain constant regionvariants to be studies in the absence of S. aureus antigen binding bythe variable domain. In one example a parental anti-RSV antibodies andan example heavy chain constant region variant (SEQ ID NO:40) werecharacterized: Shown are the amino acid sequence of an anti-RSV parentalheavy chain (SEQ ID NO:22), an example variant heavy chain (SEQ IDNO:23) and a common light chain (SEQ ID NO:24). Amino acid differencesbetween the parental antibody of allotype G1m17 and an example variantheavy chain content chain amino acid sequence are shown in boldunderlined:

Anti RSV HC parental IgG1 of allotype G1m17 HC3 (SEQ ID NO: 22)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAnti RSV variant HC of allotype G1m17 HC4 (SEQ ID NO: 23)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLSPGK Anti RSV LCLC 2 (SEQ ID NO: 24) DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

In an additional example a humanized anti-ClfA antibody and an exampleheavy chain constant region variant (SEQ ID NO: 40) were characterized.Shown are the amino acid sequence of an anti-ClfA parental heavy chain(SEQ ID NO: 25) an example variant heavy chain (SEQ ID NO:26) and acommon light chain (SEQ ID NO:27). Also shown are the variable heavy andLight chain sequences used in parental and variant antibodies (SEQ IDNO:28 and SEQ ID NO:29). Amino acid differences between the parentalantibody of allotype G1m17 and an example variant heavy chain contentchain amino acid sequence are shown in Bold underlined:

Humanized anti-ClfA HC in G1m17 heavy chain backgroundHC 5 (SEQ ID NO: 25) QVQLKESGPGLVKPSQTLSITCTISGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGNTDYNSALKSRLSISKDNSKNQVFLKMNSLTAADTAVYYCARKGEFYYGYDGFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KHumanized anti-ClfA HC in variant G1M17 heavy chain backgroundHC 6 (SEQ ID NO: 26) QVQLKESGPGLVKPSQTLSITCTISGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGNTDYNSALKSRLSISKDNSKNQVFLKMNSLTAADTAVYYCARKGEFYYGYDGFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLSPG KHumanized ClfA LC KM3 LC 3 (SEQ ID NO: 27)DIVMTQSPDSLAVSLGERVTMNCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Heavy Chain and light chain variable domain sequenced of the examplehumanized anti-ClfA antibody.

VH 5 (SEQ ID NO: 28): ClfA Humanized 12-9 VH sequenceQVQLKESGPGLVKPSQTLSITCTISGFSLSRYSVHWVRQPPGKGLEWLGMIWGGGNTDYNSALKSRLSISKDNSKNQVFLKMNSLTAADTAVYYCARKGE FYYGYDGFVYWGQGTLVTVSSVL 13 (SEQ ID NO: 29): ClfA Humanized 12-9 VL sequenceDIVMTQSPDSLAVSLGERVTMNCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCHQYLSS YTFGGGTKLEIK

Example Variant IgG1 Constant Region Sequences:

Modeling was used to investigate Immunoglobulin heavy chain constantregion interactions with a number of microbial IgBPs including SpA, Sbi,SSL10 and Protein G. Amino acids were selected from modeling studies forsubstitution in variant heavy chain constant region.

In claimed embodiments, the heavy chain constant region variant antibodyis of IgG immunoglobulin, in which at least one amino acid from theheavy chain constant region selected from the group consisting of aminoacid residues 214, 251, 252, 253, 254, 274, 276, 311, 314, 356, 358,380, 382, 384, 419, 422, 428, 431, 432, 433, 434, 435, 436 and 438 (EUnumbering) is substituted with an amino acid residue different from thatpresent in the unmodified IgG1 antibody.

The amino acid sequence of example variant IgG1 heavy chains thatattenuate the binding to one or more microbial IgBPs are shown below insequences SEQ ID NO: 30-56). The Heavy chain constant region is shownusing the one letter amino acid code (EU numbering 118-447). X denotesvariable heavy chain residues. In different immunoglobulins describedherein, the number of variable domain residues in the heavy chainvariable region may vary, where the number of X residues can be greateror less than shown in HC1-HC-27. With respect to Variant Heavy Chain FcRegion Sequences, the amino acid positions that include a residuesubstitution relative to the reference sequence of allotype G1m17,1,2are underlined. E356, M358 and A431 represent allotypic substitutionsrelative to the allotype G1m17,1,2 reference sequence (D365,L358,G431).

Heavy chain constant region 1 (SEQ ID NO: 30)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNHYTQKSLSLSPGKHeavy chain constant region 2 (SEQ ID NO: 31)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHN R YTQKSLSLSPGKHeavy chain constant region 3 (SEQ ID NO: 32)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHN R YTQKSLSLSPGKHeavy chain constant region 4 (SEQ ID NO: 33)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGN IFSCSVMHE A LHN R YTQKSLSLSPGK Heavy chain constant region 5:(SEQ ID NO: 34) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGN IFSCSVMHE A LHN R YTQKSLSLSPGK Heavy chain constant region 6:(SEQ ID NO: 35) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ EGNVFSCSVMHE A LHN R YTQKSLSLSPGK Heavy chain constant region 7(SEQ ID NO: 36) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRW QEGNVFSCSVMHEA A LHN R YTQKSLSLSPGK Heavy chain constant region 8:(SEQ ID NO: 37) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ E GN IFSCSVMHE A LHN R YTQKSLSLSPGK Heavy chain constant region 9:(SEQ ID NO: 38) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ E GN IFSCSVMHE A LHN R YTQKSLSLSPGK Heavy chain constant region 10:(SEQ ID NO: 39) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHN RF TQKSLSLSPGKHeavy chain constant region 11: (SEQ ID NO: 40)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHN RF TQKSLSLSPGKHeavy chain constant region 12: (SEQ ID NO: 41)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGN IFSCSVMHE A LHN RF TQKSLSLSPGK Heavy chain constant region 13:(SEQ ID NO: 42) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGN IFSCSVMHE A LHN RF TQKSLSLSPGK Heavy chain constant region 14:(SEQ ID NO: 43) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ EGNVFSCSVMHE A LHN RF TQKSLSLSPGK Heavy chain constant region 15:(SEQ ID NO: 44) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ EGNVFSCSVMHE A LHN RF TQKSLSLSPGK Heavy chain constant region 16:(SEQ ID NO: 45) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ E GN IFSCSVMHE A LHN RF TQKSLSLSPGK Heavy chain constant region 17:(SEQ ID NO: 46) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQ E GN IFSCSVMHE A LHN RF TQKSLSLSPGK Heavy chain constant region 18:(SEQ ID NO: 47) XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNH F TQKSLSLSPGKHeavy chain constant region 19: (SEQ ID NO: 48)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNH F TQKSLSLSPGKHeavy chain constant region 20: (SEQ ID NO: 49)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMI TRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNHYTQKSLSLSPGKHeavy chain constant region 21: (SEQ ID NO: 50)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMI TRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNHYTQKSLSLSPGKHeavy chain constant region 22: (SEQ ID NO: 51)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT L T I TRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNHYTQKSLSLSPGKHeavy chain constant region 23: (SEQ ID NO: 52)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT L T I TRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNHYTQKSLSLSPGKHeavy chain constant region 24: (SEQ ID NO: 53)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMI TRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNH F TQKSLSLSPGKHeavy chain constant region 25: (SEQ ID NO: 54)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMI TRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA A LHNH F TQKSLSLSPGKHeavy chain constant region 26: (SEQ ID NO: 55)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT L T I TRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNH F TQKSLSLSPGKHeavy chain constant region 27: (SEQ ID NO: 56)XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT L T I TRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE A LHNH F TQKSLSLSPGK

Example parental and heavy chain constant region variants have aminoacid sequences shown in SEQ ID 30-56.

In examples, parental heavy chain variable domains were combined with ahuman IgG1 heavy chain constant region of allotype G1 m17 resulting inan heavy chain constant region amino acid sequence of SEQ ID NO:30.Likewise, variable light chains were combined with a Kappa light chainconstant region of allotype Km3). Following codon optimization formammalian expression, DNA encoding a Kozak sequence, an N-terminalleader secretion sequence and the target polypeptides was synthesized,cloned into a mammalian expression vector pTT5, and expressed in HEK 293cells using methods well known in the art (described later).

Construction, Expression, Purification of Antibodies and their VariantsExample 6 Expression and Purification of Antibodies and their Variants

Antibodies and their heavy chain constant region variants were producedas follows: Codon optimization for antibody expression in 293 cells wasperformed using the OptimumGene™ Gene Design Technology (GenScript USAInc). DNA was synthesized including a 5′ EcoR1 cloning site, a Kozaksequence, and a leader signal sequence, followed by the IgG heavy orlight chain DNA sequence. The 3′ end of the Ig DNA sequences arefollowed by a stop codon and HindIII cloning site. One Synthetic DNAexamples is given in SEQ ID: 57, where XXXXXXXXXXXX represents the codonoptimized heavy or light chain DNA sequence). Oligonucleotide synthesiswas performed using methods that are well known in the art. Antibodyheavy and light chain synthetic DNA sequences were cloned into the pUC57vector using EcoR1 and Hind III cleavage sites. Plasmid preparationswere made of each plasmid and the immunoglobulin sequence inserts weresub-cloned into the expression vector pTT5 (National Research Council ofCanada (NRCC)). Plasmid preparations of immunoglobulin expressionvectors were made to provide transfection grade expression plasmids.

SEQ ID: 57 EcoR1 Kozak Sequence Leader signal peptide GAATT CGCCGCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCC XXXXXXXXXXXXTGA TAAGCTT Stop codon Hind III

Expression and Purification of Antibodies by Protein A or Protein GChromatography:

Recombinant plasmids encoding the heavy chain and light chain ofanti-microbial antibodies, or their heavy chain constant regionvariants, were transiently co-transfected into 100 mL of suspensionHEK293 cell cultures, respectively. Following confirmation of antibodyexpression, large scale HEK293 expression of antibodies was performed inbioreactors to provide 50 mg quantities of test antibodies.

HEK 293-6E cells were grown in serum free Freestyle 293 expressionmedium (Invitrogen, Carlsbad, Calif., USA). The cells were maintained inErlenmeyer Flasks at 37° C. with 5% CO2 (Corning Inc., Acton, Mass.) onan orbital shaker (VWR Scientific, Chester, Pa.). One day beforetransfection, the cells were seeded at an appropriate density in CorningErlenmeyer Flasks. On the day of transfection, DNA and PEI(Polysciences, Eppelheim, Germany) were mixed at an optimal ratio andthen added into the flask with cells ready for transfection. Thesupernatant collected on day 6 was used for purification.

Cell culture broth was centrifuged and followed by filtration. Filteredsupernatant was loaded onto a 5 mL HiTrap™ Protein G HP or HiTrap™rProtein A FF column (GE Healthcare, Uppsala, Sweden) at 1.0 mL/min.After washing and elution with appropriate buffer (The following bufferswere used affinity chromatography: Binding buffer: 20 mM PB, 150 mMNaCl, pH 7.2; Elution buffer: 50 mM citrate (pH 3.0) or 0.1M Gly-HCl (pH3.0); Neutral buffer: 1 M Tris-HCl, pH 9.0.), the fractions werecollected and neutralized with 1M Tris-HCl, pH 9.0. The purified proteinwas analyzed by SDS-PAGE Western blot by using standard protocols formolecular weight, yield and purity measurements.

Results of Expression and Purification:

As expected the anti-RSV heavy chain constant region variant (MAB5) wasnot bound by HiTrap™ rProtein A FF column. This finding confirms thatSpA no longer binds to the variant heavy chain constant region sequence(SEQ ID: 40). MAB5 was bound by the HiTrap™ Protein G HP column,demonstrating that although SpA and G both bind the CH2-CH3 interface ofIgG1, their binding involves different amino acids. Therefore, His 435and Tyr 436 are not required for binding to Protein G, as they have beenmutated to Arg 435 and Phe 436 In the variant antibody MAB5. Thus,variant antibodies having a heavy chain constant region amino acidsequence of SEQ ID: 40 do not bind to SpA via their Fc domain, but dobind to Protein G. Such antibodies can be affinity purified by Protein Gaffinity chromatography. This finding was surprising as Tyr436 makesimportant interactions with Protein G. This finding is important for theefficient purification of variant antibodies having a variant heavychain constant region containing Arg 435 and Phe 436 (all amino acidsare according to EU numbering).

Both the parental anti-SpA antibody and its variant were purified byaffinity chromatography on a HiTrap™ rProtein A FF column. Although theanti-SpA variant (MAB2) does not bind to SpA via its Fc domain (seelater; FIG. 22-26 and text), the antibody does bind SpA via its variabledomain. The purification of antibody MAB2 thus represents SpA bindingvia the variable domain of the antibody. The anti-SpA parental antibody(MAB1) can bind SpA via its Fc and variable domains.

Both the anti-ClfA parental antibody and its variant were purified byaffinity chromatography on Protein G. As was demonstrated for theanti-RSV variant antibody (MAB5), the anti-ClfA variant antibody (MAB4)also bind via its Fc domain to Protein G but not SpA. The parentalanti-ClfA was bound by both SpA and Protein G and was purified byProtein G affinity chromatography.

Characterization of Example Antibodies:

Purified antibodies were analyzed by SDS-PAGE and Western blotting. Forwestern blotting, the primary antibody was Goat-anti-Human IgG-HRP(GenScript, Cat.No:A00166).

SDS-PAGE and Western Blotting of Anti-SpA Parental Antibody MAB1 andVariant Antibody MAB2.

Anti-SpA parental antibody MAB1 (HC amino acid SEQ ID NO:19 and LC aminoacid SEQ ID NO: 21) and an example anti-SpA variant antibody MAB2 (HCamino acid SEQ ID NO:20 and LC amino acid SEQ ID NO: 21) were expressedin HEK293 cell, purified on HiTrap™ rProtein A FF, and analyzed bySDS-PAFGE and western blotting (FIG. 16).

SDS-PAGE and Western blotting of anti-Clfa parental antibody MAB3 andvariant antibody MAB4.

Anti-ClfA parental antibody MAB3 (HC amino acid SEQ ID NO:25 and LCamino acid SEQ ID NO: 27) and an example anti-ClfA variant antibody MAB4(HC amino acid SEQ ID NO:26 and LC amino acid SEQ ID NO: 27) wereexpressed in HEK293 cell, purified on Protein G, analyzed by SDS-PAGEand western blotting (FIG. 17).

Anti-RSV Control Variant Antibody (MAB5)

Anti-RSV variant antibody MAB5 (HC amino acid SEQ ID NO:23 and LC aminoacid SEQ ID NO: 24) were expressed in HEK293 cell, purified on Protein Gand analyzed by SDS-PAFG (FIG. 18). MAB5 does not bind SpA and waspurified on Protein G.

Biological Testing of Example Anti-Microbial Immunoglobulins and theirHeavy Chain Constant Region Variants Example 7 Antibody Characterizationfor Binding to S. aureus IgBP by Immuno-Diffusion

Binding to IgBPs: All procedures should be performed at room temperatureunless specified otherwise. Goat anti-mouse antibody (gamma-chainspecific, conjugated to horseradish peroxidase (HRP)) can be obtainedfrom Zymed Laboratories (Invitrogen, Inc., Carlsbad, Calif.). TMB(3,3′,5,5′-tetramethylbenzidene), a chromogenic substrate forhorseradish peroxidase enzyme activity, may be obtained from NeogenCorporation (Lansing, Mich.). The described procedures can be used forany IgBP. SpA and Sbi are used as illustrative examples. SpA may bepurchased from commercial sources or produced using standard molecularBiology methods as previously described. For example, SpA domains A, B,C, D and/or E, or their variants (Kim et al., 2010) can be PCR amplifiedusing two primers. Alternatively, the sequence of the SpA or Sbi domainor domains of interest can be synthesized and expressed using molecularbiology techniques well known in the art. PCR products can be clonedinto pET-15b to generating N-terminal His6-tagged recombinant fusionproteins. Polyhistidine-tagged fusion proteins can be purified byaffinity chromatography. Alternatively, other fusion tags such as GSTcan be used for expression and purification. Sbi or its IgFc bindingdomains were produced using standard molecular biology methods aspreviously described (Haupt et al., 2008; Zhang et al., 1999).

The following IgBPs or their domains have been used to characterize S.aureus IgBP binding to variant and parental heavy chain constant regionsequences (immune binding by the variable domain vs Fc binding by theheavy chain constant region).

Expression and Purification of SpA.

Cloning, Expression and Purification of Recombinant Sbi and SpA:

Recombinant fragments of the N-terminal region of Sbi (adjacent to thepoly-proline region) are engineered, expressed and purified as HisTagged fusions as described previously by (Burman et al. 2008, THEJOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 25, pp. 17579-17593, Jun.20, 2008). The following Sbi constructs were used in this study: Sbi-E(amino acids 28-266) containing IgG-binding domains I and II and C3interacting domains III and IV; Sbi-III/IV (amino acids 150-266). Sbifragments were purified by nickel ion-chelating chromatography.Recombinant SpA can be purchased of produced as described previously(O'Seaghdha et al., 2006).

GST-SPA-D expression: E. coli strain BL21 (DE3) was used for the GST-SpAdomain D fusion protein expression (construct provided by Prof TimothyFoster, Trinity College Dublin). Bacteria containing pGEX-KG plasmid wasgrown overnight in LB medium supplemented with 100 μg/ml ampicillin at37° C. The 15 ml primary culture was incubated with 1 L LB mediumcontaining 100 μg/ml ampicillin. Cells were grown at 37° C. with shakingat 180 rpm. The expression of the GST-SpA domain fusion protein wasinduced during the exponential phase of growth (OD₆₀₀=0.75) by addingisopropyl thiogalactoside (IPTG) to a final concentration of 0.5 mM inthe culture. E. coli cells were collected by centrifugation (8000 g, 20min) 3 h after induction. Cell pellet was suspended in 20 ml of PBS. There-suspended cells were sonicated on ice for 6 times at 80% amplitudefor 10 s separated by 10 min interval. The resulting extract wasclarified by centrifugation (60,000 g, 30 min, 4° C.). The supernatantwas collected and purified through affinity chromatography. Thesupernatant sample was split equally and loaded on a 1 ml GSTrap column(GE healthcare) using AKTA with a flow rate of 1 ml/min. The loadedcolumn was washed with 10 column volumes of PBS buffer and the boundGST-SpA domain D fusion protein was eluted with GST Elution buffer (10mM glutathione, 50 mM Tris, pH8.0). The peak fractions of the GST-SpAdomain D fusion protein was then buffer exchange into PBS buffer forfurther use.

SSL10 from S. aureus NCTC8325, kind gift from Prof Jos van Strijp and DrCarla de Haas.

Sbi-E and Sbi-III-IV Expression:

Sbi constructs were expressed in Escherichia coli strains BL21(DE3),BL21(DE3)-Star, or Rosetta (see also Burman et al. JBC 283; 17579-17593,2008). Freshly transformed E. coli cells were grown in a shaker at 37°C. in Luria Bertani broth (LB), containing ampicillin, until theyreached an extinction of 0.6 at 600 nm.Isopropyl-D-thiogalactopyranoside (Melford) was added to a finalconcentration of 0.2 mM, and the cells were incubated at 28° C. for anadditional 4 h. Cells from a 1-liter culture were harvested bycentrifugation, resuspended in 10 ml of binding buffer (20 mM Tris-HCl,0.5 M NaCl, 20 mM imidazole, pH 8.0), and lysed by sonication. Thelysate was centrifuged at 40,000 g for 15 min and the supernatantfiltered through a 0.45 μm filter. The proteins were purified usingnickel-ion chelating chromatography by either applying the filteredsupernatant to a Sartobind membrane (Sartorius) or a 1-ml HiTrap columnattached to an AKTA purifier (Amersham Biosciences). Next, the columnwas washed with binding buffer, and the bound proteins were eluted witha buffer containing 1M imidazole, for the Sartobind purification, or a0.05-1 M imidazole gradient for the HiTrap purification. Purifiedprotein was dialyzed into a buffer solution, typically 20 mM Tris, pH8.0, 100 mMNaCl, and stored at 80° C. until use.

Double Immunodiffusion Assay:

Double immunodiffusion experiments were performed on Petri dishescontaining a 1% agarose gel. Wells were punched in the agar andindividual wells filled with 50 μl of sample at 1 mg/ml in PBS (anti-SpAmonoclonals; Recombinant SpA (Biovision) GST-SpA-D; Sbi-E, Sbi III-IV orSSL10), and left to incubate for 72 h at 4° C. Insoluble proteincomplexes formed precipitin lines at the zone of equivalence. Largesoluble protein complexes were visualized by Coomassie staining.

Results: MAB1 (Parental anti-SpA antibody) formed a precipitin Line(after day 1) with SpA and (after day 2) with Sbi-E (fragment of Sbicontaining the two Ig-binding domains and two complement bindingdomains). In contract the anti-RSV variant antibody did not form aprecipitin line with either SpA or Sbi-E. As the variable domain of MAB1recognizes SpA, this result shows that binding to Sbi-E is mediated byFc binding to the parental antibody, whereas binding to SpA is likely acombination of variable domain and Fc binding. MAB2 (anti-SpA variantexample) forms a precipitin line (after day 4) only with SpA. Thisdemonstrated that MAB2, like the control variant antibody MAB5 (anti-RSVvariant antibody), does not bind via its Fc domain to Sbi-E or to SpA.The precipitin line formed with SpA represents variable domain bindingto SpA.

In a second series of immune-precipitation experiments, MAB1 (parentalanti-SpA antibody) formed a precipitin Line with SpA domain D, but notwith Sbi III/IV domains, which do not have Fc binding function. Incontact no precipitin lines are seen with variant MAB2 (panel A).Following Coomassie staining, a week precipitin line is seen with MAB2(anti-SpA variant example antibody), which represents variable domainbinding to SpA domain D. This indicates that MAB2 forms a small solublecomplex with SpA domain D, which is only visible with Coomassie stainingof the ID plate. The data show that MAB2, in contract to MAB1, does notprecipitate in the presence of the single SpA domain, but forms asoluble complex as evidenced by coomassie staining of theimmunodiffusion gel. This data is consistent with the design objectiveof the variant antibody, which has abolished the SpA and Sbi Fc bindingsites from the heavy chain constant region of the anti-SpA variantantibody (MAB2) and the anti-RSV variant antibody (MAB5).

Example 8 Antibody Characterization for Binding to S. aureus IgBP byDynamic Light Scattering (DLS)

Dynamic light scattering is a technique for measuring the size ofmolecules and nanoparticles. Scattering intensity is proportional to thesquare of the protein molecular weight, making the technique ideal foridentifying the presence of antibody antigen complexes and aggregates.DLS was used to investigate antibody antigen complex formation.

Immune complex formation was characterised by dynamic light scattering(Nano-S Zetasizer, Malvern). All readings were taken at 25° C. over athree consecutive 40-second periods in a low-volume, sealed quartzcuvette containing 50 μl samples of the anti-SPA monoclonal antibodies(1 mg/ml) and mixtures with (1 mg/ml) recombinant 4-domain SpA(Biovision); GST-SpA-D; Sbi-E, Sbi III-1V or SSL10.

FIG. 22 shows the DLS results for the control anti-RSV antibody (MAB5).The left panel shown the analysis of the control anti-RSV variantantibody alone (MAB5). The right panels show the size distribution inthe presence of either Sbi-E (fragment of Sbi containing the twoIg-binding domains and two complement binding domains) or SpA1-4 (SpAIgBP domains 1-4). There is no peak shift seen by DLS, indicate noimmune complexes have been formed between the variant antibody and theSbi or SpA IgBP domains. These results are in agreement with the lack ofprecipitin lines in ID experiments.

In contract to the variant anti-RSV antibody, the parental anti-SpAantibody (MAB1) shows a large complex pattern of peak shifts in thepresence of SpA 1-4, indicating large antibody-SpA complexes andcross-linking (FIG. 23—upper right panel)). This also occurs with Sbi-E,although the major peak shift appears relatively homogeneous, and largecomplexes are less apparent (Lower right panel-blue circle). No peakshifts were seen with SbiIII/IV (lower right panel), demonstrating thatthe interaction with Sbi is via antibody Fc interactions with the SbiFcBP domains present in Sbi-E. This result is in agreement with theresults generated by ID studies (FIGS. 20 and 21).

FIG. 24 shows the DLS analysis of the parental anti-SpA antibody (MAB1)with SpA-2 (SpA domain D alone). Peak shift DLS analysis was performedafter incubation of antibody and SpA-2 for 1 min (upper right panel) and10 mins (lower right panel). Complex formation and precipitation areseen to increase rapidly with time (FIG. 24). The results found withMAB1 are in agreement with the results generated by ID studies (FIGS. 20and 21).

In contract to the parental anti-SpA antibody, the anti-SpA variantantibody (MAB2) shows no peak shift in the presence of Sbi-E, and asmall homogeneous peak shift in the presence of SpA 1-4 (FIG. 25). Theoverlap of the MAB2 control and MAB2 in the presence of SpA 1-4 is shownin the lower left panel of FIG. 25. This result demonstrates that thevariant heavy chain constant region of MAB2 does not bind to Sbi or SpA.The complex formation seen with SpA 1-4 represents binding via thevariable domain of the anti-SpA variant antibody. Analysis of the singleSpA domain D construct (FIG. 26), did not indicate any measurable peakshift due to the small size of the single SpA domain D, and the absenceof any cross linking (FIG. 26, lower panel). The ant-SpA variantantibody (MAB2) shows a peak shift with DLS in the presence of SpA thatis consistent with a soluble complex formed via the variable domain ofthe antibody. No cross-linking or precipitation peaks can be observed.The results found with MAB2 are in agreement with the results generatedby ID studies (FIGS. 20 and 21).

Example 9 ELISA Binding to Isolated Microbial IgBPs

Such IgBP domains, variants, and IgBP domains from different S. aureusstains having a variety of amino acid substitutions within their IgBPcan be used to determine the binding to IgBP domains and full lengthproteins from different microbial stains. For example, in the case of S.aureus SpA, different domains (for example, the amino acid sequence ofSpA from clinical stains shown in FIG. 11 for domains A, D, C, D and E),can be used for binding and epitope mapping of antibodies describedherein. The antibodies that may be used in accordance with theembodiments described herein are able to bind to epitopes that block oneor more virulence functions of SpA, including Fc binding, VH3 Fabbinding vWF binding, TNFR binding, EGFR binding and osteoblast binding.Additional antibodies are able to recognize conserved functionalepitopes on SpA domains that allows the antibody to binds to multiplestains of SpA and multiple domains within such stains.

The target antigen (100 μL of 1 μg/mL SpA or Sbi antigens suspension incarbonate buffer, pH 9.2) may be coated in each well of the ELISA plates(Immulon 2; Dynex Technologies, Inc., Chantilly, Va.) for 1 hour at 37°C. After the coating step, the wells are washed twice with PBST(phosphate buffered saline (150 mM NaCl in 10 mM sodium phosphatebuffer, pH 7.4) containing 0.05% w/v Tween 20).

After discarding the last wash, coating the wells with the targetantigen, nonspecific protein-binding sites in the ELISA plates may beblocked. Two hundred microliters of PBST containing 2% (w/v) dehydratedskim milk (blotto solution) are added to each well. The plates areincubated at 37° C. for 1 hour. The blotto solution should then bediscarded. Murine IgG1 antibody or chimeric/humanized antibodies inwhich H435 of the Fc region has been mutated to R to abolish Fc bindingto SpA, (100 μL/well, diluted in wash buffer) may be added to each well.The plates are incubated for 1-2 hours at 37° C. After incubation, wellsare washed 3 times with Mild Elution Buffer pH 6.0 (Thermo Scientificcat #21033).

One hundred microliters of an appropriate dilution of Goat anti-mouse oranti-human antibody-HRP conjugate in the blotto solution may be added toeach well and incubated at 37° C. for 1-2 hours. After this incubationperiod, the conjugate solution should be removed and the wells washed 3times with PBST. After removing the last wash, 100 μL of TMB (Kblue,Neogen Cat No. 300199) can be added to each well and the plates are heldat room temperature for 1-10 minutes to observe the development of bluecolor. The relative HRP enzyme activity in each well is measured in aplate reader by absorbance of a 650-nm wavelength light source.

Example 10 Inhibition of Virulence Functions of SpA by Antibodies andtheir Variants

Inhibition of S. aureus SpA-Fc binding by anti SpA antibodies:inhibition of binding of human IgG to SpA can be tested by ELISA. ELISAplates are coated with recombinant SpA or individual domains of SpA.Purified SpA or its domains are coated onto ELISA plates in 0.1 Mcarbonate buffer, pH 9.5. Plates are incubated withperoxidase-conjugated human IgG, (The Jackson Laboratory), or purifiedlabeled human IgG1 Fc and developed using OptElA reagent. Alternatively,S. aureus cells can be used (see later method for cell ELISA). Forinhibition of labeled IgG-Fc binding, plates are incubated with anti SpAantibodies (heavy chain constant region variants are used, which do notbind to SpA via the Fc domain) before ligand binding.

Inhibition of S. aureus SpA-vWF binding by anti SpA antibodies:inhibition of binding of human IgG to SpA can be tested by ELISA. ELISAplates are coated with recombinant SpA or individual domains of SpA.Purified SpA or its variants are coated onto ELISA plates in 0.1 Mcarbonate buffer, pH 9.5. Plates are incubated withperoxidase-conjugated human vWF, (Thermo Fisher Scientific) anddeveloped using OptElA reagent. For inhibition of labeled vWF binding,plates are incubated with anti SpA antibodies (heavy chain constantregion variants are used, which do not bind to SpA via the Fc domain)before ligand binding.

Inhibition of S. aureus SpA-VH3 binding by anti SpA antibodies:inhibition of binding of human VH3 IgG to SpA can be tested by ELISA.ELISA plates are coated with recombinant SpA or individual domains ofSpA. Purified SpA or its variants were coated onto ELISA plates in 0.1 Mcarbonate buffer, pH 9.5. Plates are incubated withperoxidase-conjugated human Fab VH3, (Graille et al., 2000) anddeveloped using OptElA reagent. For inhibition of labeled VH3 Fabbinding, plates were incubated with anti SpA antibodies (heavy chainconstant region variants are used, which do not bind to SpA via the Fcdomain) before ligand binding.

Example 11 ELISA Binding to Target Microbes

Binding to S. aureus Cells.

Antibodies and their Fc variants may be tested for their ability to bindto intact cells of S. aureus. The bacterial strains used in thisexample, S. aureus can be obtained from the American Type CultureCollection (Manassass, Va.).

Bacterial cultures used for antigen preparation may be grown overnightat 37° C. in Tryptic Soy Broth. The cell suspensions are washed threetimes by centrifuging the suspension at 10,600×g for 10 minutes at 4°C., decanting the supernatant, and resuspending the pellet in 100 mMsodium bicarbonate, pH 9.5. After the final wash, the cells aresuspended in the sodium bicarbonate buffer to approximate cell densitiesof 10⁷, 10⁶, and 10⁵ colony-forming units per milliliter. Thesesuspensions can be used as antigen to coat 96-well plates. Controlsolutions, containing 1.0, 0.1, and 0.01 mg/mL, respectively, purifiedSpA are coated into several wells of each plate.

Streptavidin-conjugated alkaline phosphatase can be obtained fromJackson Immunoresearch (West Grove, Pa.) and may be diluted to a workingconcentration of 0.5 μg/mL prior to use. The alkaline phosphatasechromogenic substrate, pNPP, can be obtained from KPL (Gaithersberg,Md.). Anti-SpA monoclonal antibody SPA-27 and its correspondingbiotin-conjugated derivative may be obtained from Sigma Chemical Company(St. Louis, Mo.).

Bacterial suspensions and SpA controls may be added to a 96-well plate(100 μg/well) and the plates may be incubated at 37° C. for 1 hour. Thewells are then washed five times with PBS. Nonspecific protein-bindingsites re blocked by adding 200 L of a blotto solution (PBST with 2% w/vnonfat dehydrated milk) and the plates are held overnight at 4° C. Theplates are subsequently washed with PBST.

Unlabeled test antibody solutions may be diluted to 50 μg protein/mL inacetate buffer (500 μM NaCL/100 μM Sodium acetate, pH 3.5). Thesesolutions may be used to prepare serial 2-fold dilutions (to 0.78 μgprotein/mL) of the antibodies in acetate buffer. SPA-27 antibody is useas a positive control.

One hundred microliters of each dilution of the murine IgG1 antibodiesor chimeric/humanized antibodies of IgG1 isotype (with one or more Fcregion mutations designed to block non specific antibody binding to SpAand Sbi) are then transferred into duplicate wells and the plates areincubated at 37° C. for 1 hour. The plates may then be subsequentlywashed five times with Mild Elution Buffer pH 6.0 (Thermo Scientific cat#21033).

One hundred microliters of the diluted, biotin-conjugated anti mouse oranti human antibody may be added to the wells and the plates areincubated at 37° C. The wells may then be washed with PBST.

After washing the wells, 100 μL of streptavidin-alkaline phosphataseconjugate, diluted in blotto solution, may be added to each well and theplates may be incubated at 37° C. for 1 hour. After washing the wells,100 μL of the pNPP substrate solution is added to each well and theplates may be held at room temperature for 10 minutes. The alkalinephosphatase reaction may be stopped by adding 100 μL of 5% (w/v)disodium EDTA and the plates may be placed in a plate reader, where theabsorbance at 405-nm wavelength is read.

The IgG1 hybridoma supernatants may be diluted in sodium acetate buffer(500 μM NaCL/100 μM Sodium acetate, pH 3.5) for the binding assay. Afterthe binding reaction, the amount of antibody bound to the immobilizedbacteria is measured using the alkaline phosphatase-conjugated antibodyand detection reagents.

In an alternative method, an ELISA based screen was used to investigateanti-SpA and anti-ClfA antibody binding to S. aureus (Newman stain) anda SpA deficient S. aurues stain (ΔSpA) in the presence and absence ofhuman IgG1-Fc used to block non-specific binding and IgBP medicated Fcbinding.

ΔSpA strains of S. aureus can be generated by deletion of the spa geneon the chromosome of S. aureus Newman by allelic replacement, asdescribed previously (Bae T., and Schneewind O. (2005)).

One day before the experiment, 100 μl/well of a Staph aureus overnightculture diluted to an OD600 of 1.0 was added to a 96 well plate andincubated at 4° C. overnight. On the day of the experiment, plates werewashed with 150 μl/well PBS-T (PBS with 0.05% Tween 20) 2× then blockedwith 150 μl/well PBS-T w/0.5% BSA. The plates were agitated for 1 hourafter blocking. The plates were then washed with 150 μl/well PBS-T (2×)then 100 μl/well of primary mAb at various dilutions were added to eachELISA plate. The plate was shaken at room temp for 12 hour, washed with150 μl/well PBS-T (2×) then 100 μl/well secondary antibody (goatantihuman IgG (HRP) @ 1:5,000 in PBS-T—Thermo #31413) was added. Theplates were shaken at room temp for 1 hour, washed with 150 μl/wellPBS-T (2×) then 100 μl/well TMB was added and the plates incubated untilsufficient color change has been reached (usually around 5 minutes). 100μl/well 2M sulfuric acid was then added to stop the reaction and theplate read at OD450 on a Spectramax. In some cases, Human IgG Fc(Jackson ImmunoResearch #009-000-008) was added at 100 μg/ml to both theblocking agent and the primary antibody.

In a representative S. aureus Cell ELISA (FIG. 27), a number ofantibodies were tested for binding to S. aureus (Newman stain) and a SpAdeficient S. aureus strain (ΔSpA) in the presence and absence of humanIgG1-Fc used to block non-specific binding and IgBP medicated Fcbinding. Test Antibodies include anti-SpA MAB1 and anti-SpA variantMAB2, anti-ClfA Parental MAB, anti-RSV variant MAB5 and a non-specificanti-KLH antibody.

Discussion of ELISA Results:

The ELISA results are shown in FIG. 27. These results indicate that thecontrol (anti-KLH) and parental antibodies (non-variant antibodies) havehigh non-specific binding to S. aureus Newman stain (lower panels). Thisnon-immune binding is reduced by include human IgG1-Fc as a blocker(FIG. 27, right panels) (for example see anti-KLH and anti-ClfAantibodies). This is presumably due to blockage of IgBP Fc binding siteson the S. aureus Newman stain (FIG. 27 right panels). This is supportedby the finding that the anti-RSV variant antibody (MAB5) does not bindto S. aureus Newman stain in the absence or presence of blocking humanIgG1-Fc FIG. 27; lower panels). Thus, mutations introduced into theheavy chain constant region of variant MAB2 and MAB5 eliminate Fcbinding of the variant antibodies to S. aureus cell surface IgBPs. Incontrast, both the anti-SpA parental and anti-SpA variant antibodiesbind strongly to S. aureus Newman stain in the absence or presence ofblocking human IgG1-Fc (FIG. 27, lower panels), demonstrating variabledomain binding by the anti-SpA antibodies. The variant anti-RSV antibodyhad minimal background binding whereas the variant anti-SpA antibody hadsignificant binding.

The variant anti-SpA and anti-RSV antibodies (MAB2 and MAB5) have nobinding to SpA deficient Staph (FIG. 27, upper panels). However, Fcmediated binding of the parental anti-SpA, anti-ClfA and anti-KLHantibodies are seen that can be blocked by human IgG1-Fc (FIG. 27, Upperleft panel). This may be due to binding by alternative IgBPs expressedby the ΔSpA strain, such as S. aureus Sbi.

The results shown in FIG. 27 are tabulated in FIG. 28. These resultsindicate that the heavy chain constant region mutations in MAB2 and MAB5eliminated non-specific binding and SpA Fc medicated binding. Thespecific binding of the anti-SpA heavy chain constant region variantdemonstrated variable domain binding of this antibody to SpA.Additionally, IgG1Fc did not compete with anti-SpA antibody binding toSpA, demonstrating that the region of SpA recognized by the variabledomain of anti-SpA antibodies MAB1 and MAB2 do not overlap with theSpA-Fc binding sites.

Example 12 FACS Analysis of Antibody Binding to S. aureus

Binding of the parental anti-SpA antibody (MAB1), an example anti-SpAvariant antibody (MAB2) and a heavy chain constant region matchedanti-RSV variant control antibody (MAB6) were investigated by FACS,using Staphylococcus aureus Newman stain (FIG. 29; upper panels) or aΔSpA strain (FIG. 29, Lower panels)) grown in log phase (FIG. 29, leftpanels), or from stationary phase cultures (FIG. 29; right panels).Standard FACS methods known in the art were used for the analysis. Thesecond antibody used in the study was Alexa Fluor 488 conjugated Fabgoat anti-human IgG. As can be see in FIG. 29, the control anti-RSVvariant antibody did not bind to S. aureus under all conditions tested,confirming that the heavy chain constant region variant chain does notbind to S. aureus FcBPs via its Fc domain. This confirms the data seenin cellular ELISA assays. In contrast, the parental and variant anti-SpAantibodies (MAB1 and MAB2) bound strongly to S. aureus from stationaryor log phase cultures, but not to the ΔSpA strain of S. aureus. Thisresult confirms that antibody binding by the variant anti-SpA antibody(MAB2) is mediated by the variable domain of the antibody.

Effector Function Testing of Variant Antibodies

In a variety of in vivo and in vitro settings, antibody coating oftargets has been shown to mediate potent killing mechanisms such ascomplement-dependent cytotoxicity (CDC), antibody-dependent cellularcytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) andopsonophagocytosis. These effector functions are mediated by theantibody heavy chain constant region. To verify that variant antibodiesdescribed herein do not have attenuated effector function, due to theintroduction of mutations that attenuate bacterial IgBP binding,antibodies can be tested in a number of binding assays (FcγRs and C1qbinding) and effector function assays (Complement depositionopsonophagocytosis, CDC, ADCC, ADCP, anti-microbial activity),

Construction, Expression, and Purification of FcγRs: FcγR binding of Fcvariant antibodies: FcγRs can be constructed as C-terminal-6×His-GSTfusions, expressed in 293T (human FcγRs) cells, and purified by usingnickel affinity chromatography. Detailed methods are provided in Lazaret al., 2006.

FcγR binding of parental and Fc variant antibodies: Variants areconstructed, expressed and purified, and can be screened for FcγRaffinity by using an AlphaScreen assay. AlphaScreen assays can useuntagged variant IgG1 to compete the interaction between biotinylatedIgG bound to streptavidin donor beads and FcγR-His-GST bound to anti-GSTacceptor beads.

True binding constants can be obtained by a competition surface plasmonresonance (SPR) experiment. Competition SPR experiments measured captureof free Ab from a preequilibrated Ab/receptor analyte mixture to V158FcγRIIIa-His-GST bound to an immobilized anti-GST surface. Equilibriumdissociation constants (K_(D) values) are calculated by using theproportionality of initial binding rate on free Ab concentration in theAb/receptor equilibrium. Detailed description of AlphaScreen and SPRassays is provided in Lazar et al., 2006 and references therein. SPRmeasurements were performed using a BIACore 3000 instrument (GEHealthcare). Fcγ R affinity can be determined as described in Nieba etal., 1996.

C1q binding of parent and variant antibodies: Surface plasmon resonancedetermination of binding affinities. SPR measurements can be performedin HBS-EP running buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% v/v surfactant P20, GE Healthcare) using a BIACore 3000instrument (GE Healthcare).

For determining C1q affinity of IgG1κ antibodies and their variants, aProtein L CM5 biosensor chip (GE Healthcare) can be generated using astandard primary amine coupling protocol. The chip's reference channelcan be coupled to bovine serum albumin (BSA) to minimize nonspecificbinding of C1q. Antibodies at 50 nM can be immobilized on the protein Lsurface for 0.5 or 1 min at 10 μL/min. C1q in 2-fold serial dilutions(starting at 100 or 25 nM, 5 concentrations total) is injected overantibody-bound surface for 3 min at 30 μL/min followed by a 4.5 mindissociation phase. C1q molarity can be calculated using the molecularweight of the C1q hexameric bundle, 410 kDa. After each cycle, thesurface can be regenerated by injecting glycine buffer (10 mM, pH 1.5).In order to subtract nonspecific C1q binding to antibody-coated proteinL surface, an Fc variant with greatly ablated CDC activity can beincluded. Sensorgrams can be processed by zeroing time and responsebefore the injection of C1q and by subtracting appropriate nonspecificsignals (response of BSA-blocked reference channel, response of an Fcvariant with ablated CDC, and response of running buffer). Kineticparameters can be determined by global fitting of association anddissociation phase data with a two-state binding model (A+B AB AB*).K_(d) was calculated as K_(d1)/(1+1/K_(d2)).

ADCC of Parent and Fc Variant Antibodies.

ADCC can be measured by using either the DELFIA EuTDA-based cytotoxicityassay (PerkinElmer) or the LDH Cytotoxicity Detection Kit (RocheDiagnostics). Human PBMCs can be purified from leukopacks by using aFicoll gradient and allotyped for V/F158 FcγRIIIa by using PCR. NK cellscan be isolated from human PBMCs by using negative selection andmagnetic beads (Miltenyi Biotec, Auburn, Calif.). Target cell lines canbe obtained from American Type Culture Collection. For Eu-baseddetection, target cells are first loaded with BATDA[Bis(acetoxymethyl)-2,2′:6′,2″-terpyridine-6,6″-dicarboxylate] at 1×10⁶cells per ml and washed 4×. For both Eu- and LDH-based detection, targetcells can be seeded into 96-well plates at 10,000 cells per well andopsonized by using Fc variant or WT Abs at the indicated finalconcentration. Triton X-100 and PBMCs alone can be run as controls.Effector cells can be added at 25:1 PBMCs:target cells or 4:1 NKcells:target cells, and the plate are incubated at 37° C. for 4 h. Cellsare incubated with either Eu³⁺ solution or LDH reaction mixture, andfluorescence can be measured by using a Fusion Alpha-FP (PerkinElmer).Data can be normalized to maximal (Triton) and minimal (PBMCs alone)lysis and fit to a sigmoidal dose-response model.

ADCP of Parent and Fc Variant Antibodies.

For phagocytosis experiments, monocytes can be isolated from humanV/F158 FcγRIIIa PBMCs by using a Percoll gradient and differentiatedinto macrophages by culture with 0.1 ng/ml granulocyte/macrophagecolony-stimulating factor for 1 week. For quantitative ADCP, targetcells (e.g. WIL2-S for anti CD20 antibody Fc variants) can be labeledwith PKH67, seeded in a 96-well plate at 20,000 cells per well, andtreated with WT or variant Ab at the designated final concentrations.Macrophages are labeled with PKH26 (Sigma) and added to the opsonizedlabeled target cells at 20,000 cells per well, and the cells areco-cultured for 18 hours. Fluorescence is measured by using dual-labelflow cytometry.

CDC of parental and Fc variant antibodies can be tested initially in thecontext of an anti CD20 antibody as described in Moore et al., 2010. ForCDC assays, target Ramos or Raji cells can be washed 2× in RHB Buffer(RPMI Medium 1640 containing 20 mM HEPES, 2 mM glutamine, 0.1% BSA, pH7.2) by centrifugation and resuspension and seeded at 40,000 cells perwell. Native IgG1 or variant antibody is added at the indicated finalconcentrations. Human serum complement (Quidel, San Diego, Calif.) arediluted with RHB buffer and added to opsonized target cells. Plates canbe incubated for 2 hr at 37° C., Alamar Blue is added, cells arecultured overnight, and fluorescence is measured in relativefluorescence units. Data is normalized to maximal (Triton X-100) andminimal (complement alone) lysis and fitted to a sigmoidal dose-responsecurve.

FcRn binding of variant anti-SpA antibodies: FcRn binding can bemeasured as described previously (Dall'Acqua et al., 2006; Datta-Mannanet al., 2006).

Anti-S. aureus effector function can be tested in a number of in vitroassays. These assays may include a C1q deposition, C3 deposition,bacterial opsonophagocytic assays and bactericidal assay, which aredescribed below.

Example 13 Anti-S. aureus C1q Deposition Assays of Selected Antibodiesand their Fc Variants

C1q Deposition Assay:

This assays tests for the ability of antibodies to deposit complement onbacteria. Add 100 μl of bacteria (@ OD600 1.0) to microtubes washing 1×with 1 ml HBSS+, Centrifuge at ˜7000×g (9000 rpm), for 5 minutes at 4°C. Next, add 50 μl I of a 2× concentration of test antibody or isotypecontrol diluted in GV buffer. Add 50 μl of human complement @ 20%diluted in GV buffer for a 10% final concentration. Incubate samples at37° C. in shaking water bath for 60 minutes, then wash 2× with 1 mlHBSS+, ˜7000×g (9000 rpm), for 5 minutes at 4° C. Add 100 μl of a mouseanti-human C1q mAb, or C1q isotype control for a final concentration of3 μg/ml in GV buffer, incubate for 30 minutes @ 4° C. Next, wash 2× with1 ml of HBSS+].

FACS detection of complement on bacteria: Add 100 μl of anti-mouseIgG-PE at a 1:50 dilution in HBSS+ at 4° C. Incubate for 30 minutes, inthe dark, on ice with shaking. Wash 2× with 1 ml HBSS, ˜7000×g (9000rpms), for 5 minutes at 4° C. Resuspend in 0.5 ml of HBSS+, at 4° C.Transfer samples to FACS tubes. Analyze samples by Accuri gating onbacteria, 10,000 events, FL2.

Reagents: HBSS+: w/Mg Ca. #14025-092, Gibco; Gelatin veronal buffer(GV), #G6514, Sigma; Human Serum Complement #A113, Quidel—(thaw rapidlyin 37° C. water bath to ˜90% leaving small pellet, mix and put on ice,aliquot and store at −80° C.); Mouse-IgG1 anti-human C1q antibody (1.1mg/ml), #A201, Quidel; Negative control for anti-human C1q Mab: Anti-TNPmouse IgG1 (isotype control for C1q mAb), NA/LE, clone 107.3, stock=1.0mg/ml, #554721, BD Pharmingen; PE-conjugated F(ab′)2 fragment donkeyanti-mouse IgG (H+L) antibody, #715-116-150, Jackson ImmunoResearch—(rehydrate with 1.0 ml distilled water and add 20 μl of stockper 980 μl of HBSS+=1:50 dilution); Distilled water, #15230, Gibco.

Results: C1q deposition assays were performed to test whether theparental anti-SpA (MAB1), its variant anti-SpA (MAB2), and the controlanti-RSV variant (MAB5) antibodies are able to can deposit C1q on wildtype S. aureus Newman and a S. aureus ΔSpA strain (FIG. 30). A dosetitration of the test antibodies was performed using S. aureus WT andΔSpA Newman strain in the presence of pooled human serum as a source ofcomplement. As shown in FIG. 30a , the variant anti-SpA antibody (MAB2),deposits C1q on the surface of the wild type S. aureus Newman strain ina dose dependent manner, while the parental anti-SpA antibody (MAB1) andnegative control anti-RSV variant antibodies (MAB5) lack this function(FIG. 30a , upper panel). The ability of the anti-SpA variant antibody(MAB2) to deposit C1q on S. aureus is lost in assays using the ΔSpA S.aureus stain, which has no SpA expression (FIG. 30b ). This resultdemonstrates that the anti-SpA variant antibody shows antigen dependentdeposition of complement on the S. aureus Newman strain. Thisdemonstrates that FcBPs expressed by S. aureus are able to neutralizethe C1q effector function of the parental IgG1 antibodies, but not thatof its variants such as MAB2. The FACS data from FIG. 30 is tabulated inFIG. 31.

Example 14 C3 Complement Deposition Assay

C3 deposition was determined using S. aureus stain JE2 and measured byFACS. The following methods were used. Staph JE2 was grown overnight inTHB at 37° C. with shaking. Next day, stationary phase culture werewashed and resuspended in PBS to OD_(600 nm)=0.4. Aliquot 1 ml ofbacterial culture into eppendorf tubes and spin at max speed for 2 min,then resuspend the pellet in 50 μl of HEPES buffer (120 mM HEPES, 140 mMNaCl, 5 mM CaCl₂ and 25 mM MgCl₂). Dilute pooled human serum to 10% inHEPES buffer. Add 50 μl of 10% serum to the bacteria.

Add anti-SpA parental (MAB1) or variant antibodies (MAB2) at a finalconcentration of 2 μg/ml. Incubate for 30 min at 37° C. Spin max/2 min,Wash 1× with 1 ml of 0.1% BSA+PBS, then resuspend in 100 ul of αC3b(diluted 1:200 in 0.1% BSA+PBS (Protos Immuno Research)). Incubate for20 min at 4° C. and then wash as described above. Resuspend in 500 μl ofPBS and analyse using FACS (FIG. 32).

Results: As can be seen in FIG. 32, the anti-SpA parental antibody wasunable to deposited C3 on the surface of S. aureus JE2 (Control-black vsMAB1—Red). In contract, the anti-SpA variant antibody resulted in strongC3 deposition of the surface of S. aureus JE2 (MAB2-green). This resultreinforces that data seen for C1q deposition, and demonstrates that S.aureus interacts with the heavy chain constant region of parentalantibodies, blocking their effector function. This interaction ispresumably mediated by S. aureus IgBPs including SpA. In contract,variant antibody MAB2 maintains its effector function as demonstrated byrobust C1q and C3 deposition on the surface of S. aureus SpA expressingstains.

Example 15 Neutrophil-Mediated Opsonophagocytic Assay

An opsonization assay may be a colorimetric assay, a chemiluminescentassay, a fluorescent or radiolabel uptake assay, a cell-mediatedbactericidal assay, or any other appropriate assay known in the artwhich measures the opsonic potential of a substance and therebyidentifies reactive immunoglobulin. In an opsonization assay, aninfectious agent, a cell, and the opsonizing substance to be tested areincubated together.

In certain embodiments, the opsonization assay is a cell-mediatedbactericidal assay. In this in vitro assay, an infectious agent such asa bacterium, a phagocytic cell, and an opsonizing substance such asimmunoglobulin, may be incubated together. Any eukaryotic cell withphagocytic or binding ability may be used in a cell-mediatedbactericidal assay. In certain embodiments, phagocytic cells aremacrophages, monocytes, neutrophils, or any combination of these cells.Complement proteins may be included to promote opsonization by both theclassical and alternate pathways.

In one method, the ability of parental and variant anti-SpA antibodiesand control antibodies were evaluated for the ability of example testantibodies to mediate the phagocytosis of opsonized bacteria labeledwith FITC.

The following method was performed: Resuspend FITC labeled bacteria in 1ml cold OPA buffer (HBSS Ca++& Mg+++0.2% BSA) at ˜4.0E+08 CFU/ml.Opsonize with specific antibodies or control. Add 100 μl of Mab in OPAbuffer to bacteria-FITC pellet (see above) for 30 minutes, 37 C shakingwater bath. Aspirate dry, keep on ice in dark until phagocytosis assayset up. Add 100 μl of washed PMNs at 10E+06/ml (1.0E+06/tube) toopsonized bacterial cell pellet in mirotube, transfer to 12×75 mmpolypropylene FACS tube. Incubate in 37° C. shaking H2O bath for 30minutes. Next, add 100 μl of cold quench/tube (to quench the staining ofany externally bound bacteria) vortex, add 2 ml of cold AB. Spin for 5minutes at 1,200 rpm, 4° C. Decant supernatant and wash again with 2 mlof AB, 4° C. Add 0.5 ml of AB/tube (4° C.) read on Accuri, collect 5000events, FL1. Reagents: OPA buffer: HBSS Ca++& Mg+++0.2% BSA; AB:Dulbecco's DPBS-+2% FBS: Quench: Trypan blue (Gibco #15250-061) diluted1:3 in DPBS- (1 ml trypan blue and 2 ml PBS): HBSS: Mg++Ca++, Gibco,#14025-092; DPBS: no Mg++Ca++, Sigma # D8537 or Lonza/BioWhittaker#17-512Q

Phagocytosis Results: The anti-SpA parental antibody (MAB1) and anexample variant anti-SpA antibody (MAB2) were tested in two phagocytosisassay (FIG. 33 and FIG. 34). In the first assay (FIG. 33), two controlantibodies were used (an anti-RSV variant (MAB5) and a non-specificparental anti-KLH antibody). S. aureus Newman stain and a ΔSpA strainlacking SpA expression were used at the target bacteria. As shown inFIG. 33, the anti-SpA variant antibody was able to enhance thephagocytosis of the S. aureus wild type Newman strain as compared tocontrol antibodies. The control antibodies were able to induce somenon-specific uptake. The parental anti-SpA antibody gave a similarresults as the control antibodies, demonstrating that S. aureus is ableto suppress the effector function of the parental ant-SpA antibody(MAB1), but not that of its variant (MAB2). No enhancement ofphagocytosis was seen using the ΔSpA S. aureus strain, demonstratingvariable domain specificity of the enhanced effector function of theanti-SpA variant antibody MAB2.

In a second opsono-phagocytosis assay format, anti-SpA MAB 1 and 2(variant) were tested (FIG. 34). The opsonic ability of an antibody isdetermined by the amount or number of infectious agents remaining afterincubation. The fewer the number of infectious agents that remain afterincubation, the greater the opsonic activity of the antibody tested. Ina cell-mediated bactericidal assay, opsonic activity is measured bycomparing the number of surviving bacteria between two similar assays,only one of which contains the antibody being tested. Alternatively,opsonic activity is determined by measuring the number of viableorganisms before and after incubation with a sample antibody. A reducednumber of bacteria after incubation in the presence of antibodyindicates a positive opsonizing activity. In the cell-mediatedbactericidal assay, positive opsonization is determined by culturing theincubation mixture under appropriate bacterial growth conditions. Anyreduction in the number of viable bacteria comparing pre-incubation andpost-incubation samples, or between samples, which containimmunoglobulin, and those that do not, is a positive reaction. As can beseen (FIG. 34) the variant anti-SpA antibody (MAB2) resulted insignificant enhanced opssonphagocytic activity as measured by bacterialsurvival when compared to the Parental MAB1. The control representsbacterial survival in the absence of added antibody.

Example 16 Neutrophil-Mediated Opsonophagocytic Bactericidal Assay

Opsono-phagocytic killing of S. aureus JE2 using pooled human serum. Thefollowing assay was used to test parental and variant anti-SpAantibodies for their effect on the opsonophagocytic killing of S. aureusJE2

Bacteria were grown overnight in THB. In the morning dilute cultures1:40 in fresh THB and grow to OD_(600 nm)=0.4. Pellet S. aureus at @4000 rpm for 10 min then wash in 10 ml of PBS. Centrifuge as above andresuspend in 300 ul of PBS. Adjust to the OD_(600 nm)=0.4 in 3 ml ofPBS. Dilute bacteria 1:5 in pooled human serum using siliconized tube.Test antibodies (MAB1 and MAB2) were added to tubes at a finalconcentration of 5 μg/ml and 25 μg/ml. Tubes were incubated at 37° C.for 30 min, then diluted 1:40 in RPMI. 100 μl of bacteria were added to100 ul of neutrophils (MOI=0.5) in 96 well tissue culture plates, spunat 1600 rpm for 5 min and incubate at 37° C.+CO₂ for 30 min. After 30min, serial dilutions were made in molecular grade water and then platedon THA plates.

Results: It can be seen from FIG. 35 that there was a significantincrease in opsonophagocytic killing of S. aureus JE2 by the variantanti-SpA antibody as compared to the parental anti-Spa antibody.

Example 17 Generation of Additional Humanized Anti-SpA IgG1 Antibodiesand their Variants

Additional Anti SpA Antibodies:

In additional examples of anti-SpA Fc variant antibody design, CDRsequences from murine antibodies were used for the design of additionalpreferred anti-SpA humanized antibodies. These parental humanizedantibodies were then used for the design of Fc variant antibodies aspreviously described in Example 5. The heavy and light chain CDR aminoacid sequences from antibodies 3F6, 5A10 and 3D11 (Kim et al., 2012)were obtained from Patent Application WO 2013/142349 A1. In oneembodiment, 3F6 is selected for generating an anti-SpA antibody becauseit was able to bind to all 5 domains of SpA and to Sbi (Kim et al.,2012). In another embodiment, antibody 5A10 is selected for generatingan anti-SpA antibody because it was able to recognize all 5 SpA domains,but not Sbi. To design chimeric and humanized antibodies using thelimited sequence data available, CDR sequences from each antibody werefirst used to design murine heavy and light chain variable domainsequences based on CDR amino acid sequence alignment. Designed sequencesare shown in SEQ ID NO:58-63 below. In SEQ ID NO 59, 61 and 63, (X)represents an insertion of 0, 1 or 2 amino acids).

SEQ ID NO: 58 (LC CDR murine graft-GKV4-5501-IGKJ1):QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPPTFGGG TKLEIK SEQ ID NO: 59(HC CDR murine graf-IGHV5-9-4*01-IGHJ4):EVQLVESGGGLVKPGGSLKLSCAASGFAFSNYDMSWVRQSPEKRLEWVAEISSGGTYPYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCAR(X)GGFLITTRDYYAMDYWGQGTSVTVSS SEQ ID NO: 60(LC CDR murine graft-IGKV3-1*01-IGKJ1):DIVLTQSPASLAVSLGQRATISCRASESVEYSGASLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPS TFGGGTKLEIKSEQ ID NO: 61 (HC CDR murine graft-IGHV10S3*01-IGHJ4):EVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYC(X)VTEHYDYDYYVMDYWGQGTSVTVSS SEQ ID NO: 62(LC CDR murine graft-IGKV4-8601-IGKJ1):EIVLTQSPAITAASLGQKVTITCSASSSVSYMHWYQQKSGTSPKPWIYEISKLASGVPARFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSYPFTFGSGT KLEIK SEQ ID NO: 63(HC CDR murine graft-IGHV1S30*01-IGHJ4):EVQLQQSGPELVKLGPSVKISCKASGYSFTSYYMHWVKQSHGKSLEWIGEIDPFNGGTSYNQKFKGKATLTVDTSSSTAYMELHSLTSEDSLVYYCAR(X)YGYDGTFYAMDYWGQGTSVTVSS

The murine heavy (SEQ ID NO 59, 61 and 63) and light chain variablesequences (SEQ ID NO 58, 60 and 62) were then used for the design ofchimeric antibodies (containing murine variable domain sequences andhuman constant domain sequences). Such methods can be used for any heavyand light chain combination selected from SEQ ID NO: 58 to 63. Examplesof the construction of a chimeric antibodies and their Fc variants areprovided, using the CDR grafted murine variable region sequences SEQ IDNO:60 (light chain variable domain) and SEQ ID NO:61 (heavy chainvariable domain). The murine IGHV10-1 gene (SEQ ID NO: 70) was used togenerate the CDR grafted murine heavy chain (SEQ ID NO: 61). This isthen combined with a human IgG1 heavy chain constant sequence ofallotype G1m17 (SEQ ID NO:30). As described previously, any IgG1allotype can also be used for the IgG heavy chain construction.

The heavy chain amino acid sequence of the resulting Fc chimericantibodies are provided as SEQ ID NO 71 and 72. Likewise, the murinevariable light chain sequence mIGKV3-1 (SEQ ID NO: 64) was used togenerate a CDR grafted variable murine Kappa light chain and full lengthlight chain (SEQ ID NO 60 and 65).

Fc variant antibodies can be constructed as described previously usingFc variants provided in SEQ ID NO: 31-56. For example, variant heavychains incorporating the Fc region of SEQ ID NO: 40 are provided (SEQ IDNO: 73-74).

Following codon optimization of the target polypeptides for mammalianexpression, DNA encoding a Kozak sequence, an N-terminal leadersecretion sequence can be synthesized, cloned into a mammalianexpression vector such as pTT5, and expressed in HEK 293 cells usingmethods well known in the art (for example, as described previouslyherein).

murine VL sequence mIGKV3-1: SEQ ID NO: 64DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYChimeric light chain amino acid sequence mIGKV3-cdr graft: SEQ ID NO: 65DIVLTQSPASLAVSLGQRATISCRASESVEYSGASLMQWYQQKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHuman VL sequence IGKV1D-39*1: SEQ ID NO: 66DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPHuman VL sequence IGKV4-1*1: SEQ ID NO: 67DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST PHumanized light chain amino acid sequencehIGKV1D-39-cdr graft-IGKJ1-hIgKC: SEQ ID NO: 68DIQMTQSPSSLSASVGDRVTITCRASESVEYSGASLMQWYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRKVPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHumanized light chain amino acid sequencehIGKV4-1-cdr graft-IGKJ1-hIgKC: SEQ ID NO: 69DIVMTQSPDSLAVSLGERATINCRASESVEYSGASLMQWYQQKPGQPPKLLIYAASNVESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKVPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECmurine VH sequence IGHV10-1: SEQ ID NO: 70EVQLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVRChimeric heavy chain amino acid sequence mIGHV10-IGHJ4-hIgG1SEQ ID NO: 71 EVQLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKChimeric heavy chain amino acid sequence mIGHV10-IGHJ4-hIgG1SEQ ID NO: 72 EVQLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCARVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKChimeric heavy chain variant amino acid sequence mIGHV10-IGHJ4-hIgG1SEQ ID NO: 73 EVQLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLS PGKChimeric heavy chain variant amino acid sequence mIGHV10-IGHJ4-hIgG1SEQ ID NO: 74 EVQLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSESMLYLQMNNLKTEDTAMYYCARVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLS LSPGKHuman VH sequence IGHV3-73 SEQ ID NO: 75EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQAPGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRHuman VH sequence IGHV3-23_1 SEQ ID NO: 76EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHumanized heavy chain amino acid sequence: hIGHV3-73graft-IGHJ4-hIgG1SEQ ID NO: 77 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKHumanized heavy chain amino acid sequence: hIGHV3-73graft-IGHJ4-hIgG1SEQ ID NO: 78 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKHumanized heavy chain amino acid sequence: hIGHV3-23graft-IGHJ4-hIgG1SEQ ID NO: 79 EVQLLESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVSRIRSKSNNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKHumanized heavy chain amino acid sequence: hIGHV3-23graft-IGHJ4-hIgG1SEQ ID NO: 80 EVQLLESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVSRIRSKSNNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

CDR Grafting and Humanization of Anti-SpA Antibodies:

CDR grafting can be used to humanize murine antibodies sequences.Designed murine variable domain sequences SEQ ID NOs:58-63 were used toBLAST human IGHV and IGKV germline sequences using methods known in theart. The closest human V-gene alignments to sequences SEQ ID NOs:58-63were used for the design of CDR grafted humanized heavy and lightchains. A number of V genes with varying degrees of amino acid identityto the search sequence can be used for humanization. Examples of humangerm line IGLV and IGHV gene sequences selected following alignment tomurine SEQ ID NO: 58-63 are as follows: SEQ ID NO: 58: IGKV3-11*1; SEQID NO: 59: IGHV3-66*4; SEQ ID NO: 60: IGKV4-1*1 and IGKV1D-39*1; SEQ IDNO: 61: IGHV3-73*2, IGHV 3-73*1 and IGHV 3-23*1; SEQ ID NO: 62:IGKV3-11*1; SEQ ID NO: 63: IGHV1-46*3. In addition to the aboveexamples, any other human V gene sequence or allotype can be used forCDR grafting.

Examples of the construction of CDR grafted, humanized anti-SpAantibodies and their Fc variants are provided. Such grafted humanizedantibodies sequences, in addition to an affinity maturation process mayrequire an additional maturation process, resulting in one or morematuration mutations to arrive at a therapeutic humanized antibodyhaving optimal affinity and other improved physiochemical propertiessuch as affinity, avidity, stability, solubility, expression level,and/or biological activity. Standard methodology known to thosepracticing the art can be used for both CRD grafting, affinitymaturation or physicochemical optimization. In one illustrative example,CDR grafting was used to humanize the sequence of an anti-SpA murineantibody using the HC and LC CDR sequences from DNA encoding theantibody 3F6. The same methods can be used for the humanization ofantibodies derived from the murine CDR grafted sequences SEQ ID NO: 58,59, 62 and 63. As a non-limiting example of the humanization method,designed murine variable LC and HC sequences SEQ ID 60 and HC 61 areshown. The closest human germ line V-gene alignments to the murine VLSEQ ID NO: 60 and VH sequence SEQ ID NO 61 were used for the design ofCDR grafted humanized heavy and light chains. For the construction ofCDR grafted light and heavy chains, the following human germ linesequences were selected for grafting IGKV4-1*1 (SEQ ID NO:67),IGKV1D-39*1 (SEQ ID NO:66), IGHV3-73, (SEQ ID NO:75) and IGHV 3-23*1(SEQID NO:76).

CDRs sequences were grafted into human IgG1 heavy chain and Kappa lightantibody backbone sequences. The resulting humanized parental heavy (SEQID NOs:77-80) and light chain (SEQ ID NOs:68 and 69) sequences areprovided. The IgG1 allotype of the example provided it that on G1m17(SEQ ID NO:30). As described previously, any IgG1 allotype can also beused for the IgG heavy chain construction.

Following codon optimization of the target polypeptides for mammalianexpression, DNA encoding a Kozak sequence, an N-terminal leadersecretion sequence can be synthesized, cloned into a mammalianexpression vector such as pTT5, and expressed in HEK 293 cells usingmethods well known in the art (as described herein). Heavy chains (SEQID NOs: 77-80) can be expressed with either light chain (SEQ ID NOs: 68or 69). The resulting secreted antibody can be purified from culturemedia using methods described previously or known in the art, examplesof which are described herein.

For the construction of humanized Fc variants derived from the describedparental heavy chains (SEQ ID NOs: 77-80), Fc domain variant sequencescan be used as described for Example 5. Examples of such substitutevariant heavy chain constant sequences are provided in SEQ ID NOs:31-56, and anti-SpA humanized Fc variant heavy chains using onesubstitute variant heavy chain constant sequence (SEQ ID NO:40) areshown in SEQ ID NOs:81-84 below.

Humanized variant heavy chain amino acidsequence: hIGHV3-73graft-IGHJ4-hIgG1: SEQ ID NO: 81EVQLVESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAREHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLS PGKHumanized variant heavy chain amino acidsequence: hIGHV3-73graft-IGHJ4-hIgG1: SEQ ID NO: 82EVQLVESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVGRIRSKSNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLS LSPGKHumanized variant heavy chain amino acidsequence: hIGHV3-23graft-IGHJ4-hIgG1: SEQ ID NO: 83EVQLLESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVSRIRSKSNNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLS PGKHumanized variant heavy chain amino acidsequence: hIGHV3-23graft-IGHJ4-hIgG1: SEQ ID NO: 84EVQLLESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGLEWVSRIRSKSNNYATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVTEHYDYDYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLS LSPGK

Fc variant antibodies derived from SEQ ID NOs:77-80 can be constructedas described previously using Fc variants provided in SEQ ID NOs:31-56.For example variant heavy chains incorporating the Fc region of SEQ IDNO:40 are provided (SEQ ID NOs:81-84). Following codon optimization ofthe target polypeptides (i.e. heavy chain, or its variant and lightchain) for mammalian expression, DNA encoding a Kozak sequence, anN-terminal leader secretion sequence can be synthesized, cloned into amammalian expression vector such as pTT5, and expressed in HEK 293 cellsusing methods well known in the art (examples of which are describedherein). Heavy chains (SEQ ID No: 81-84) can be expressed with eitherdescribed light chain (SEQ ID No: 68 or 69). The resulting secretedantibody can be purified from culture media using methods describedpreviously or known in the art.

Heavy chain constant region Fc variants which do not bind SpA arepreferred for used in screening chimeric, CDR grafted and affinitymutated antibodies so as to avoid SpA-Fc binding in ELISA assays, and toallow accurate binding measurements to be made with full lengthantibodies using ELISA, BIACore or DLS (Dynamic Light Scattering).

Following codon optimization for mammalian expression, DNA encoding aKozak sequence, an N-terminal leader secretion sequence and the targetpolypeptides are synthesized, cloned into a mammalian expression vectorpTT5, and expressed in HEK 293 cells using methods well known in the art(described previously). Methods previously described in in Examples 5 to16 can be used for expression, purification and biological analysis ofparental or variant anti-SpA antibodies.

In another example of the generation of a humanized anti-SpA antibodiesand their variants, the CDRs from murine antibody 5A10 was used for thegeneration of chimeric and humanized antibodies as described above.Designed murine variable LC and HC sequences are shown in SEQ ID NOs:58and 59. The closest human germ line V-gene alignments to the murine VLSEQ ID NO: 58 and VH sequence SEQ ID NO 59 were IGKV3-11*1 andIGHV3-66*4. These germ line VL and LH sequences were used for the designof CDR grafted humanized heavy and light chains.

CDRs sequences were grafted into human IgG1 heavy chain and Kappa lightantibody backbone sequences. The resulting humanized parental heavy (SEQID No: 86-87) and light chain (SEQ ID NO:85) sequences are provided. TheIgG1 allotype of the example provided it that on G1m17 (SEQ ID NO:30).As described previously, any IgG1 allotype can also be used for the IgGheavy chain construction. Additionally, other germ line human VH and VLsequences can be used for CDR grafting.

Humanized light chain amino acid sequenceIGKV3-11*-1-cdr graft-IGKJ1-hIgKC: SEQ ID NO: 85EIVLTQSPATLSLSPGERATLSCRASQSSVSYLAWYQQKPGQAPRLLIYDTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSYPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECHumanized heavy chain amino acid sequence:hIGHV3-66*4 graft-IGHJ4-hIgG1: SEQ ID NO: 86EVQLVESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPGKGLEWVSVISSGGTYPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARARGGFLITTRDYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKHumanized heavy chain amino acid sequence:hIGHV3-66*4 graft-IGHJ4-hIgG1: SEQ ID NO: 87EVQLVESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPGKGLEWVSVISSGGTYPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGFLITTRDYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

For the construction of humanized Fc variants derived from the describedparental heavy chains (SEQ ID No: 86-87), Fc domain variant sequencescan be used as described for Example 5. Examples of such substitutevariant heavy chain constant sequences are provided in SEQ ID NO: 88-89.

Humanized variant heavy chain amino acidsequence: hIGHV3-66*4 -23graft-IGHJ4-hIgG1: SEQ ID NO: 88EVQLVESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPGKGLEWVSVISSGGTYPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARARGGFLITTRDYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLS LSPGKHumanized variant heavy chain amino acidsequence: hIGHV3-66*4 graft-IGHJ4-hIgG1: SEQ ID NO: 89EVQLVESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPGKGLEWVSVISSGGTYPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGFLITTRDYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV Q FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN RF TQKSLSLS PGK

Following codon optimization of the target polypeptides for mammalianexpression, DNA encoding a Kozak sequence, an N-terminal leadersecretion sequence can be synthesized, cloned into a mammalianexpression vector such as pTT5, and expressed in HEK 293 cells usingmethods well known in the art (as described herein). Heavy chains (SEQID No: 86-89) can be expressed with light chain (SEQ ID No: 85). Theresulting secreted antibody can be purified from culture media usingmethods described previously or known in the art, examples of which aredescribed herein.

Example 18 Attenuation of Superantigen Type Binding of VH3 DerivedHumanized Antibodies to SpA

Some of the claimed humanized antibodies of the current inventionutilize heavy chain variable sequences derived from the IGHV-3 family ofgerm line V gene sequences. Antibodies using such IGHV-3 gene sequenceshave the potential to bind SpA in a superantigen like manner (asdescribed in Graille et al., 2000). The IGHV amino acids responsive forsuch non-immune binding have been defined from the crystal structure ofS. aureus Protein A complexed with the Fab fragment of a human VH3derived antibody.

Interactions involve the following heavy chain amino acids: H15, H17,H19, H57, H59, H64, H65, H66, H68, H69, H70, H80, H81, H82a and H82b(FIGS. 3a and b ). To attenuate superantigen binding to IGHV-3 sequencespresent in the variable region of the heavy chains of any of theantibodies of the invention described herein, one or more amino acidchanges can be introduced at contact residues (i.e. one or more changescan be introduced at positions selected from the list including but notlimited to H15 G, H17 S, H19R, H57 X (X can be K, I or T), H59 Y, H64 K,H65 G, H66 R, H68 T, H70 S, H81 Q, H82a N and H82b S) such that theresulting amino acid is different from that of the original parentalIGHV-3 derived VH gene sequence. In other words, according to someembodiments, a variant immunoglobulin heavy chain that is part of ananti-Staphylococcus aureus (e.g., anti-SpA) variable heavy chainsequence variant antibody includes one or more amino acid substitutionsin its variable heavy chain sequence as compared to a parental anti-SpAantibody, wherein the one or more amino acid substitutions include oneor more Kabat positions selected from heavy chain positions H15, H17,H19, H57, H59, H64, H65, H66, H68, H69, H70, H80, H81 and, H82a, H82b.The parental antibody may be any suitable anti-Staphylococcus aureus oranti-SpA antibody including, but not limited to, an anti-SpA humanizedantibody, an anti-SpA Fc variant antibody, an anti-SpA matured antibody,or an anti-SpA matured Fc variant antibody

Using the X-ray structure (FIG. 3), which defines IGHV-3 SpAinteractions, and the IMGT, NCBI and VBASE databases of germ line IGHV3alleles/polymorphisms, the following amino acid substitutions weredesigned to attenuate SpA IGHVH3 interactions:

H19R to K (found in 3-73)

H82a N to I (found in 3-15*08)

H82a N to S (found in 3-30*15)

H80/N82a to L to V/N to S (found in 3-64*3 and *5)

H81 Q to H ((found in 3-47*01)

H68 T to A ((found in 3-30*09)

H68/H69 TI to NT ((found in 3-25*1 to *5)

H68 Y to H ((found in 3-63*1 and *2)

H17 S to A ((found in 3-13*02)

The above amino acid changes can be introduced either alone or in anycombination so as to attenuate SpA-VH3 binding.

In a second approach to selection of amino acid substitutions atpositions including but not limited to H15 G, H17 S, H19 R, H57 X (X canbe K, I or T), H59 Y, H64 K, H65 G, H66 R, H68 T, H70 S, H81 Q, H82a Nand H82b S, analysis of in vivo somatic hyper mutation events wereanalyzed using data in the NCBI archive of antibody sequences. IGHVmutations are aligned to IGHV-3 germ line sequences as described inBowers et al 2013, (THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO.11, pp. 7688-7696, Mar. 15, 2013). Mutations were then modeled onto thex-ray crystal structure of the SpA IGHV-3 interface (FIG. 3) andmutations predicted to disrupt SpA finding to IGHV-3 germ line sequencesinclude, but are not limited to:

H175 to P

H19R to G. K or T

H57K, I or T to A, P, R, or S

H59Y to F, H, N, or S

H68 T to A, I or S

H70S to F

H81Q to E, H or R

H82a N or G to D, H, K, S, T

H82b S to G, N, or T

Such amino acid changes can be introduced either alone or in anycombination so as to attenuate SpA-VH3 binding. Such mutations can beintroduced into antibodies VH domains of the invention by methods knownin the art.

Alternatively, the antibody can be modified by in vitro or in vivo SHMso as to introduce mutations into the variable domain, includingframework regions, that attenuates IGHV-3 superantigen binding to SpA,but are neutral, or enhance affinity of the variable domain for antigenspecific binding to Spa or Sbi.

REFERENCES

The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entirety, as if fully set forthherein.

-   Acharya K R, Passalacqua E F, Jones E Y, Harlos K, Staurt D I, Brehm    R D. Structural basis of superantigen action inferred from crystal    structure of toxic-shock syndrome toxin-1. Nature 1994; 367: 94-7;-   Almagro J C, Fransson J (2008) Humanization of antibodies. Front    Biosci 13:1619-1633-   Al-Shangiti A M, Nair S P, Chain B M (2005) Clin Exp Immunol    140:461-469-   ANDERS LARSSON* AND JOHN SJOQUIST, JOURNAL OF CLINICAL MICROBIOLOGY,    December 1989, p. 2856-2857 Vol. 27-   Arcus V L, Langley R, Proft T, Fraser J D, Baker E N (2002a) J Biol    Chem 277:32274-32281.-   Arcus V (2002b) Curr Opin Struct Biol 12:794-801-   Ashkenazi et al., 1997, Curr Opin Irnrnunol 9: 195-200,-   Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, Oguchi A, Nagai Y,    Iwama N, Asano K, Naimi T, et al. (2002) Lancet 359:1819-1827-   Baca et al., 1997, J. Biol. Chern. 272(16):10678-10684;-   Barnes. N, and P. Mark Hogarth Bruce D. Wines, Maree S. Powell,    Paul, The Journal of Immunology, 2000, 164: 5313-5318.-   Bassler, B. L. (1999) Curr. Opin. Microbiol. 2, 582-587-   J, G., Beavis, R. C.& Novick, R. P. (1995) Proc. Natl. Acad. Sci.    USA 92, 12055-12059-   Benito, Y., Kolb, F. A., Romby, P., Lina, G., Etienne, J. &    Vandenesch, F. (2000) RNA 6, 668-679-   Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc.    Natl. Acad. Sci. U.S.A. 85:58795883-   Bjorck (1988) Protein L. A novel bacterial cell wall protein with    affinity for Ig L chains. The Journal of Immunology, Vol 140, Issue    4: 1194-1197-   Bjorck, L., and Kronvall, G. (1984) J. Immunol. 133,969-974,-   Bohach, G. A., Fast, D. J., Nelson, R. D. & Schlievert, P. M. (1990)    Crit. Rev. Microbiol. 17, 251-272.-   Bouma, B., de Groot, P. G., van den Elsen, J. M., Ravelli, R. B.,    Schouten, A., Simmelink, M. J., Derksen, R. H., Kroon, J., and    Gros, P. (1999) EMBO J. 18, 5166-5174-   Bowers et al., PNAS Dec. 20, 2011 vol. 108 no. 51 20455-20460-   Boyle, M. D. P. (1990) in Bacterial Immunoglobulin-Binding Proteins,    ed. Boyle, M. P. D. (Academic, San Diego), Vol. 1, pp. 17-28,-   Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458,-   Burman et al., JBC. 283, 25, 17579-17593, 2008, Itoh et al., Mol    Immunol 2010 January; 47(4):932-8.-   Burman, J. D., Leung, E., Atkins, K. L., O'Sheaghdha, M. N., Lango,    L., Bernardo, P., Bagby, S., Svergun, D. I., Foster, T. J.,    Isenman, D. E., and van den Elsen, J. M. H. (2008) J. Biol. Chem.    283, 17579-17593-   Burmeister W P, Huber A H, Bjorkman P J (1994) Nature 372:379-383)-   Burton, D. R. (1985) Mol. Immunol. 22, 161-206;-   Cary, S., Krishnan, M. R., Marion, T. & Silverman, G. J. (1999) Mol.    Immunol. 36, 769-776)-   Casadevall A E, Dadachova E, Pirofski L A. Passive antibody therapy    for infectious diseases. Nat Rev Microbiol 2004; 2(9): 695-703-   Cary, S., Lee, J., Wagenknecht, R. & Silverman, G. J. (2000) J.    Immunol. 164, 4730-4741)-   Chamow et al., 1996, Trends Biotechnol 14:52-60;-   Cheng, A. G., H. K. Kim, M. L. Burts, T. Krausz, O. Schneewind,    and D. M. Missiakas. 2009. Genetic requirements for Staphylococcus    aureus abscess formation and persistence in host tissues. FASEB J.    23:3393-3404. doi:10.1096/fj.09-135467-   Cheung, A. L. & Projan, S. J. (1994) J. Bacteriol. 176, 4168-4172;-   Chien, Y. & Cheung, A. L. (1998) J. Biol. Chem. 273, 2645-2652-   Clark, 2000, Immunol Today 21:397-402,-   Clark, 1997, IgG effector mechanisms, Chern Immunol. 14 Oct. 19,    2006 65:88-110;-   Clark, E., Upadhyay, A., Bagby, S., and van den Elsen, J. (2009)    Mol. Immunol., 46, 2834-2835.-   Claro T, Widaa A, O'Seaghdha M, Miajlovic H, Foster T J, et    al. (2011) Staphylococcus aureus Protein A Binds to Osteoblasts and    Triggers Signals That Weaken Bone in Osteomyelitis. PLoS ONE 6(4):    e18748, 2011-   Dall'acqua, W., Johnson, L. S., Ward, E. S.: US20070122403A1 (2007)-   Davies et al., 2001, Bioteehnol Bioeng 74:288-294-   Datta-Mannan et al (2006) Drug Metabolism and Disposition 35, 86-94-   Deisenhofer, 1981, Biochemistry 20:2361-2370-   De Jonge M, Burchfield D, Bloom B, et al. Clinical trial of safety    and efficacy of inh-a21 for the prevention of nosocomial    Staphylococcal bloodstream infection in premature infants. J.    Pediatr 2007; 151(3): 260-265-   De Lano, W. L., Ultsch, M. H., De Vos, A. M., and    Wells, J. A. (2000) Science 287, 1279-1283-   De Pascalis et al., 2002, J. Immunol. 169:3076-3084-   Derrick, J. P. and Wigley, D. B. (1992) Nature (London) 359,    752-754;-   Domanski et al., INFECTION AND IMMUNITY, August 2005, p. 5229-5232-   Emsley J., Cruz M., Handin R. et. al. Crystal structure of the von    Willebrand Factor A1 domain and implications for the bind ing of    platelet glycoprotein Ib. J Biol. Chem. 273: 10396-10401, 24 Apr.    1998.-   Fagan et al., INFECT. IMMUN 1991, 69, 851-4857-   Firan et al., Int Immunol. 2001 13:993-1002,-   Forsgren & Sjouist, J Immunol. 1966; 97:822-7-   Foster T. J., Nature Rev. Immunol, 3, 2005, 948-958 and references    cited therein.-   Foster, T. J., O'Reilly, M., Patel, A. H. & Bramley, A. J. (1988)    Antonie Van Leeuwenhoek, 54, 475-482. 17. Patel, A. H., Nowlan, P.,    Weavers, E. D. & Foster, T. (1987) Infect. Immun. 55, 3103-3110.-   Furatawa et al., 1975, Keller et al., J Immunol. 1976:772-7; Sprague    et al., J. Virol., Apr. 1, 2008; 82: 3490-3499, Lilley et al., J    Virol 2001 75, 11218-   Garman et al., 2000, Nature 406:259-266-   Gemmell et al., J. Med. Microbiol.—Vol. 46 (1997), 208-2 13;-   Gemmell et al., 1991 Zentralbl Bakteriol (Suppl.), 273-277-   Ghetie et al., 2000, Annu Rev Immunol 18:739-766;-   Gorman & Clark, 1990, Semin Immunol 2(6):457-66-   Gomez, M. I., et al. 2004. Nat. Med. 10:842-848-   Gomez, M. I., Seaghdha, M. O., and Prince, A. S. 2007. EMBO J.    26:701-709.-   Gomez M I, O'Seaghdha M, Magargee M, Foster T J, Prince A S (2006)    Staphylococcus aureus protein A activates TNFR1 signaling through    conserved IgG binding domains. J Biol Chem 281: 20190-20196.-   Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185;-   Gouda, H., Torigoe, H., Saito, A., Sato, M., Arata, Y., and    Shimada, I. (1992) Biochemistry 31, 9665-9672-   Gouda, H., Shiraishi, M., Takahashi, H., Kato, K., Torigoe, H.,    Arata, Y., and Goward, C., Scawen, M., Murphy, J. and    Atkinson, T. (1993) Trends Biochem. Sci. 18, 136-140,-   Goodyear, C. S., and Silverman, G. J. (2004) Proc. Natl. Acad. Sci.    U.S.A. 101, 11392-11397-   Griffiths et al., 1998, Curr Opin Biotechnol 9: !O2108,-   Graille et al., Proc Natl Acad Sci USA. 2000 May 9; 97(10):    5399-5404-   Hall et al., INFECTION AND IMMUNITY, December 2003, p. 6864-6870-   Haupt K, Reuter M, van den Elsen J, Burman J, Ha{umlaut over (    )}lbich S, et al. (2008) The Staphylococcus aureus Protein Sbi Acts    as a Complement Inhibitor and Forms aTripartite Complex with Host    Complement Factor H and C3b. PLoS Pathog 4(12): e1000250.    doi:10.1371/journal.ppat.1000250-   Hayhurst & Georgiou, 2001, Curr Opin Chern Biol 5:683-689;-   Haynes B F, Fauci A S. Introduction to immune system. In: Braunwald    E, Fauci A S, Kasper D L, Hauser S L, Longo D L, Jameson J L,    editors. Harrison's principles of internal medicine. New York:    McGraw Hill; 2005. pp. 1907-30)-   He et al., 1998, J. Immunol. 160: 1029-1035;-   Herr A B, Ballister E R, Bjorkman P J (2003) Nature 423:614-620-   Heden, L.-O., Frithz, E., and Lindahl, G. (1991) Eur. J. Immunol.    21, 1481-149-   Hillson, J. L., Karr, N. S., Oppliger, I. R., Mannik, M. &    Sasso, E. H. (1993) J. Exp. Med. 178, 331-336.)-   Hoogenboom H R (2005) Selecting and screening recombinant antibody    libraries. Nat Biotechnol 23:1105-1116.-   Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449    domain. Science. 297: 1176-1179, 16 Aug. 2002.-   Herr A B, Ballister E R, Bjorkman P J (2003) Nature    423:614-620 (2000) Infect Immun 68:4407-4415-   Huizinga E. G., Tsuji S., Romijn R. A. et. al. Structures of    glycoprotein Ibalpha and its complex with von Willebrand factor A1    domain. Science. 297: 1176-1179, 16 Aug. 2002-   Hulstein J. J., de Groot P. G., Silence K. et. al. A novel nanobody    that detects the gain-of-function phenotype of von Willebrand factor    in ADAMTS13 deficiency and von Willebrand disease type 2B. Blood.    106: 3035-3042, 1 Nov. 2005.-   Huizinga E. G., Tsuji S., Romijn R. A. et. al. Structures of    glycoprotein Ibalpha and its complex with von Willebrand factor A1-   Idusogie et al., 2000, J Immunol 164:4178-4184-   James L C, Keeble A H, Khan Z, Rhodes D A, Trowsdale J (2007) Proc    Natl Acad Sci USA 104:6200-6205)-   Jansson, B., Uhlen, M., and Nygren, P. A. (1998) FEMS Immunol. Med.    Microbiol. 20, 69-78-   Jefferis, R and LeFranc, M-P, 2009, mABs 1: 1-6,-   Jefferis et al., 2002, Immunol Lett 82:57-65-   Jerlstro et al., (1991) Mol. Microbiol. 5, 843-849;-   Jones et al., 1986, Nature 321:522-525;-   Kazeeval T. N. and A. B. Shevelev, Biochemistry (Moscow), 2009, Vol.    74, pp. 12-21;-   Kim et al., 2001, J. Mol. Evol. 54:1-9, hereby entirely incorporated    by reference Kim H et al. JEM 2010; 207:1863-1870-   Kim H. K, Emolo, C., DeDent, A. C, Falugi, F., Dominique M.    Missiakas, D. M., and Schneewind, O. (2012) Infect Immun. 80,    3460-70-   Kotzin, B. L., Leung, D. Y., Kappler, J. & Marrack, P. (1993) Adv.    Immunol. 54, 99-166.-   Kozlowski, L. M., Kunning, S. R., Zheng, Y., Wheatley, L. M. &    Levinson, A. I. (1995) J. Clin. Immunol. 15, 145-151-   Krapp et al., 2003, J Mol Bioi 325:979-989-   Kristiansen, S. V., Pascual, V. & Lipsky, P. E. (1994) J. Immunol.    153, 2974-2982.-   Kroll M. H., Hellums J. D., McIntire L. V. et. al. Platelets and    shear stress. Blood. 88: 1525-1541, 1 Sep. 1996.-   Krauss et al., 2003, Protein Engineering 16(10):753-759.-   Kronvall, J Immunol. 1973 November; 111(5):1401-6; Schroder et al.,    1986 Immunology, 57, 305-   Langley R, Wines B, Willoughby N, Basu I, Proft T, Fraser J D (2005)    JMonteiro R C, Van De Winkel J G (2003) Annu Rev Immunol 21:177-204)-   Lazar G A, Dang W, Karki S, et al. Engineered antibody    immunoglobulin variants with enhanced effector function. Proc Natl    Acad Sci USA 2006; 103(11): 4005-4010.-   Lehner, T, Monoclonal antibodies against microorganisms. Curr Opin    Immunol 1989; 1(3): 462-466;-   Levinson, A. I., L. Kozlowski, Y. Zheng, and L. M. Wheatley. 1995. B    cell superantigens: definition and potential impact on the immune    response. J. Clin. Immunol. 15:26S-36S;-   Levinson, A. I., and L. Kozlowski. 1996. Staphylococcal protein A:    functional properties of a model B-cell superantigen, p. 99-106.    In M. Zouali (ed.), Human B-cell superantigens. Landes Bioscience    Publishers, Austin, Tex.-   Lewis M J, Pleass R J, Batten M R, Atkin J D, Woof J M (2005) J    Immunol 175:6694-670)-   Lewis, M. J., Meehan, M., Owen, P., and Woof, J. M. JBC. 283,    17615-17623, 2008;-   Meehan, M., Lynagh, Y., Woods, C., and Owen, P. (2001) Microbiology    147, 3311-3322-   Li, H., Llera, A., Malchiodi, E. L. & Mariuzza, R. A. (1999) Annu.    Rev Immunol. 17, 435-466.-   Little et al., 2000, Immunol Today 21:364-370-   Lina G, Bohach G A, Nair S P, Hiramatsu K, Jouvin-Marche E, Mariuzza    R (2004) J Infect Dis 189:2334-2336;-   Llewely, M and Cohen, J Lancet Infectious Diseases 2, Issue 3    156-162, 2002;-   Loghem, E., Frangione, B., Recht, B., and Franklin, E. C. (1982)    Scand. J. Immunol. 15, 275-278, 21-   Loghem Evan, 1986, Allotypic markers, Monogr Allergy 19: 40-51-   Lonberg N (2005) Human antibodies from transgenic animals. Nat    Biotechnol 23:1117-1125-   Maillard et al., JBC, 2004, 279, pp. 2430-2437, 2004-   Marasco W A, Sui J, The growth and potential of human antiviral    monoclonal antibody therapeutics. Nat Biotechnol 2007 25(12):    1421-1434;-   Mascari L. M. and Ross J. M. Quantification of    staphylococcal-collagen binding interactions in whole blood by use    of a confocal microscopy shear-adhesion assay. J Infect. Dis. 188:    98-107, 1 Jul. 2003.-   Martin et al., 2001, Mol Cell 7:867-877-   Martin et al., J. C J. Clin. Invest. 119:1931-1939 (2009)-   Maynard & Georgiou, 2000, Annu Rev Biorned Eng 2:33976,-   Maxwell et al., 1999, Nat Struct Bioi 6:437-442-   Mimura et al., 2001, J Bioi Chern 276:45539-45547-   Moks, T., Abrahmsen, L., Nilsson, B., Hellman, U., Sjoquist, J., and    Uhlen, M. (1986) Eur. J. Biochem. 156, 637-643-   Moore, G. L., Chen, H., Karki S., and Lazar, G. A. mAbs (2010) 2,    181-189-   Morea et al., 1997, Biophys Chem 68:9-16;-   Morea et al., 2000, Methods 20:267279-   Morfeldt, E., Taylor, D., von Gabain, A & Arvidson, S. (1995) EMBOJ.    14, 4569-4577.-   Nardella et al., Mol Immunol. 1985 June; 22(6):705-713-   Nardella F A, et al., J Immunol. 1987 Feb. 1; 138(3):922-92-   Nieba, L., Krebber, A. & Pluckthun, A. (1996) Anal. Biochem. 234,    155-165.-   Nilson, B. H., et al. (1993). J. Immunol. Methods 164, 33-40-   Nizet, J Allergy Clin Immunol 2007; 120:13-22-   Novak L., Deckmyn H., Damjanovich S. et. al. Shear-dependent    morphology of von Willebrand factor bound to immobilized collagen.    Blood. 99: 2070-2076, 15 Mar. 2002.-   Novick R. P. Mol Microbiol. 2003 June; 48(6):1429-49-   Novick, R. P., Ross, H. F., Projan, S. J., Kornblum, J.,    Kreiswirth, B. & Moghazeh, S. (1993) EMBO J. 12, 3967-3975;-   O'Connor et al., 1998, Protein Eng 11:321-8.-   Ogata & Shigeta, Infect Immun. 1979; 770-4.-   O'Seaghdha M, van Schooten C J, Kerrigan S W, Emsley J, Silverman G    J, et al. (2006) Staphylococcus aureus protein A binding to von    Willebrand factor A1 domain is mediated by conserved IgG binding    regions. FEBS J 273: 4831-4841-   O'Toole, P., L. Stenberg, M. Rissler, and G. Lindahl. Proc Natl Acad    Sci USA. 1992 89: 8661-8665;-   Palmqvist N, Foster T, Tarkowski A, Josefsson E. Protein A is a    virulence factor in S. aureus arthritis and septic death. Microb    Pathog 2002; 33: 239-49-   Papageorgiou A C, Acharya K R. Microbial superantigens: from    structure to function. Trends Microbiol 2000; 8: 369-75;-   Papageorgiou, A. C., Tranter, H. S. & Acharya, K. R. (1998) J. Mol.    Biol. 277, 61-79-   Para, Goldstein & Spear, J Virol. 1982; 41, 137-44-   Patel et al., J. Immunol. 2010; 184; 6283-6292-   Pawar P., Shin P. K., Mousa S. A. et. al. Fluid shear regulates the    kinetics and receptor specificity of Staphylococcus aureus binding    to activated platelets. J Immunol. 173: 1258-1265, 15 Jul. 2004.-   Peng, H. L., Novick, R. P., Kreiswirth, B., Kornblum, J. &    Schlievert, P. (1988) J. Bacteriol. 170, 4365-4372)-   Pleass R J, Dunlop J I, Anderson C M, Woof J M (1999) J Biol Chem    274:23508-23514, Carayannopoulos L, Hexham J M, Capra J D (1996) J    Exp Med 183:1579-1586)-   Pleass R J, Areschoug T, Lindahl G, Woof J M (2001) J Biol Chem    276:8197-8204.)-   Presta L G. Molecular engineering and design of therapeutic    antibodies. Curr Opin Immunol 2008; 20(4): 460-470]-   Presta et al., 1997, Cancer Res. 57(20):4593-9;-   Provenza G, Provenzano M, Visai L, Burke F M, Geoghegan J A,    Stravalaci M, Gobbi M, Mazzini G, Arciola C R, Foster T J,    Speziale P. FEBS J. 2010 November; 277(21):4490-505.-   Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;-   Radaev et al., 2001, J Bioi Chem 276:16469-16477-   Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915;-   Raghavan et al., 1996, Annu Rev Cell Dev Bioi 12:181-220-   Ramsland, P. A., Willoughby, N., Trist, H. M., Farrugia, W.,    Hogarth, P. M., Fraser, J. D., and Wines, B. D. (2007) Proc. Natl.    Aacd. Sci. U.S.A. 104, 15051-15056;-   Ravetch et al., 2001, Annu Rev Immunol 19:275-290-   Recht, B., Frangione, B., Franklin, E., and van Loghem, E. (1982) J.    Immunol. 127-   Recsei, P., Kreiswirth, B., O'Reilly, M., Schlievert, P., Gruss A. &    Novick, R. P. (1986) Mol. Gen Genet. 202, 58-61;-   Reddy S. T et al., Nature Biotechnology 28, 965-969 (2010)-   Reiter et al., 1996, Nature Biotech. 14:12391245-   Reis, K. J, Ayouh, E. M, and Boyle, M. D. P. (1984) J. Immunol. 132    3091-3097-   Riechmann et al., 1988; Nature 332:323-329;-   Roben, P. W., Salem, A. N., and Silverman, G. J. (1995) J. Immunol.    154, 6437-6445-   Roque et al., 2004, Biotechnol. Prog. 20:639-654-   Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969973-   Romagnani, S., Giudizi, M. G., del Prete, G., Maggi, E., Biagiotti,    R., Almerigogna, F. & Ricci, M (1982) J. Immunol. 129, 596-602)-   Rosok et al., 1996, J. Biol. Chern. 271(37): 22611-22618;-   Rupp M E, Holley H P, Lutz J, et al. Phase ii, randomized,    multicenter, double-blind, placebo-controlled trial of a polyclonal    anti-S. aureus capsular polysaccharide immune globulin in treatment    of S. aureus bacteremia. Antimicrob Agents Chemother 2007; 51(12):    4249-4254.-   Sadler J. E. Biochemistry and genetics of von Willebrand factor.    Annu. Rev Biochem. 67: 395-424, 1998.-   Sasso, E. H., Silverman, G. J. & Mannik, M. (1989) J. Immunol. 142,    2778-2783.-   Sasano, M., Burton, D. R. & Silverman, G. J. (1993) J. Immunol. 151,    5822-5839.-   Sauer-Eriksson et al., 1995, Structure 3:265-278-   Seppala, I., Kaartinen, M., Ibrahim, S. & Makela, O. (1990) J.    Immunol. 145, 2989-2993.-   Shields et al., 2001, J Bioi Chern 276:6591-6604-   Shields et al., 2002, J Bioi Chern 277:26733-26740-   Shimada, I. (1998) Biochemistry 37, 129-136-   Sidorin, E. V. and Solov'eva, T. F. Biochemistry (Moscow), 2011,    Vol. 76, 295-308-   Silverman et al., J Exp Med. 2000 Jul. 3; 192(1):87-98;-   Siedlecki C. A., Lestini B. J., Kottke-Marchant K. K. et. al.    Shear-dependent changes in the three-dimensional structure of human    von Willebrand factor. Blood. 88: 2939-2950, 15 Oct. 1996.-   Silverman, G. J. 1997. B cell superantigens. Immunol. Today    18:379-386.-   Silverman, G. J., Nayak, J. V., Warnatz, K., Najjar, F. F., Cary,    S., Tighe, H. & Curtiss, V. E. (1998) J. Immunol. 161, 5720-5732.-   Simmons et al., 2002, J Irnrnunol Methods 263:133-147-   Sondermann et al., 2001, J Mol Biol 309:737749-   Sondermann et al., 1999, Embo J 18:1095-1103-   Sondermann et al., 2000, Nature 406:267-273-   Sprague E R, Wang C, Baker D, Bjorkman P J (2006) PLoS Biol 4:e148.)-   Starovasnik, M. A., O'Connell, M. P., Fairbrother, W. J. &    Kelley, R. F. (1999) Protein Sci. 8, 1423-1431)-   Starovasnik, M. A., Skelton, N. J., O'Connell, M. P., Kelley, R. F.,    Reilly, D., and Fairbrother, W. J. (1996) Biochemistry 35,    15558-15569-   Tan et al., 2002, J. Immunol. 169:1119-1125;-   Tashiro, M. & Montelione, G. T. (1995) Curr. Opin. Struct. Biol. 5,    471-481-   Tashiro et al., 1995, Curr Struet Bioi 5:471-481-   Torpier, Capron & Ouaissi, Nature 1979 278, 447-9-   Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies,    Molecular Biology of B Cells, 533-545, Elsevier Science (USA)-   Uhlen et al., 1984, J Biol Chem 259, 1695-1702-   Uff S., Clemetson J. M., Harrison T. et. al. Crystal structure of    the platelet glycoprotein Ib(alpha)N-terminal domain reveals an    unmasking mechanism for receptor activation. J Biol. Chem. 277:    35657-35663, 20 Sep. 2002.-   Umaiia et al., 1999, Nat Bioteehnol 17:176-180-   Viau, M., Longo, N. S., Lipsky, P. E., and Zouali, M. (2005) J.    Immunol. 175, 7719-7727-   Vaccaro C, Zhou J, Ober R J, Ward E S. Engineering the fc region of    immunoglobulin g to modulate in vivo antibody levels. Nat Biotechnol    2005; 23(10): 1283-1238;-   van Egmond M, van Garderen E, van Spriel A B, Damen C A, van    Amersfoort E S, van Zandbergen G, van Hattum J, Kuiper J, van de    Winkel J G (2000) Nat Med 6:680-685-   van Egmond M, Damen C A, van Spriel A B, Vidarsson G, van Garderen    E, van de Winkel J G (2001) Trends Immunol 22:205-211. 43.-   Verhoeyen et al., 1988, Science, 239:1534-1536;-   Vidarsson, et al., BLOOD, 15 Nov. 2006 VOLUME 108, NUMBER 10).    Relative to the wild-type antibody, the H435A mutant is deficient in    transfer across the placenta-   Weems J J, Steinberg J P, Filler S, et al. Phase ii, randomized,    double-blind, multicenter study comparing the safety and    pharmacokinetics of tefibazumab to placebo for treatment of S.    aureus bacteremia. Antimicrob Agents Chemother 2006; 50(8):    2751-2755.-   Watkins J. F. (1964) Nature, 202, 1364; Chapman T. L. et al., JBC    1999, 274, 6911-   Williams R J, Ward J M, Henderson B, Poole S, O'Hara B P, Wilson M,    Nair S P (2000) Infect Immun 68:4407-4415-   Wines, B. D., Willoughby, N., Fraser, J. D., and    Hogarth, P. M. (2006) J. Biol. Chem. 281, 1389-1393-   Wines B D, Willoughby N, Fraser J D, Hogarth P M (2006) J Biol Chem    281:1389-1393. 24.-   Wrammert. J, Smith. K, Miller. J, Langley. W. A, Kokko. K, Larsen,    C, Zheng, N.Y., Mays. I, Garma. L, Helms. C, James. J, Air. G. M,    Capra. J. D, Ahme. R, & Wilson P. C. Nature. 2008 May 29; 453(7195):    667-671.-   Woof J M (2002) Biochem Soc Trans 30:491-494.,-   Wu et al., 1999, J. Mol. Biol. 294:151-162;-   Xiong J. P., Stehle T., Goodman S. L. et. al. New insights into the    structural basis of integrin activation. Blood. 102: 1155-1159, 2003-   Yee et al., Virology. 1982; 120:376-82-   Zhang, L., Jacobsson, K., Vasi, J., Lindberg, M., and    Frykberg, L. (1998) Microbiology 144, 985-991)-   Zeitlin L, Cone R A, Moench T R, Whaley K J. Preventing infectious    disease with passive immunization. Microbes Infect 2000; 2(6):    701-708

1-28. (canceled)
 29. A humanized anti-SpA antibody comprising animmunoglobulin heavy chain which comprises a sequence selected from SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; and an immunoglobulinlight chain which comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, or SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
 30. The humanizedanti-SpA antibody of claim 29, wherein the variable light chainsequence, the variable heavy chain sequence, or both, further compriseone or more maturation mutations resulting in one or more amino acidsubstitutions, deletions, or insertions that improve one or moreproperties of the humanized anti-SpA antibody selected from affinity,avidity, stability, solubility, expression level, and/or biologicalactivity.
 31. A humanized anti-SpA antibody comprising an immunoglobulinheavy chain which comprises a variable heavy chain sequence and aconstant heavy chain sequence, wherein the immunoglobulin heavy chaincomprises SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ IDNO:86, or SEQ ID NO:87; and an immunoglobulin light chain whichcomprises a variable light chain sequence and a constant light chainsequence.
 32. The humanized anti-SpA antibody of claim 31, wherein thevariable light chain sequence, the variable heavy chain sequence, orboth, further comprise one or more maturation mutations resulting in oneor more amino acid substitutions, deletions, or insertions that improveone or more properties of the humanized anti-SpA antibody selected fromaffinity, avidity, stability, solubility, expression level, and/orbiological activity.
 33. The humanized anti-SpA antibody of claim 31,wherein the immunoglobulin light chain comprises SEQ ID NO:68, SEQ IDNO:69, or SEQ ID NO:85.
 34. The humanized anti-SpA antibody of claim 33,wherein the variable light chain sequence, the variable heavy chainsequence, or both, further comprise one or more maturation mutationsresulting in one or more amino acid substitutions, deletions, orinsertions that improve one or more properties of the humanized anti-SpAantibody selected from affinity, avidity, stability, solubility,expression level, and/or biological activity.
 35. The humanized anti-SpAantibody of claim 34, wherein the immunoglobulin heavy chain comprisesone or more amino acid substitutions in its constant heavy chainsequence as compared to that of a parental anti-SpA antibody, resultingin an anti-SpA variant antibody.
 36. The anti-SpA variant antibody ofclaim 35, wherein the anti-SpA variant antibody is an anti-SpA Fcvariant antibody; and wherein the parental anti-SpA antibody is thehumanized anti-SpA antibody of claim 10, the humanized anti-SpA antibodyof claim 11, the humanized anti-SpA antibody of claim 12, the humanizedanti-SpA antibody of claim 13, the humanized anti-SpA antibody of claim14, or the humanized anti-SpA antibody of claim
 15. 37. The anti-SpAvariant antibody of claim 35, wherein the anti-SpA variant antibody isan anti-SpA Fc variant antibody; and wherein the heavy chain constantdomain comprises an amino acid sequence selected from SEQ ID NOs: 31-56.38. The anti-SpA variant antibody of claim 37, wherein the anti-SpAvariant antibody is an anti-SpA Fc variant antibody; and wherein thevariant immunoglobulin heavy chain comprises SEQ ID NO:81, SEQ ID NO:82,SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:88, or SEQ ID NO:89.
 39. Theanti-SpA variant antibody of claim 35, wherein the variable light chainsequence, the variable heavy chain sequence, or both, further compriseone or more maturation mutations resulting in one or more amino acidsubstitutions, deletions, or insertions that improve one or moreproperties of the anti-SpA variant antibody selected from affinity,avidity, stability, solubility, expression level, and/or biologicalactivity.
 40. The anti-SpA variant antibody of claim 31, having at leastone additional amino acid substitution which results in alteration of anIgG1 allotype, and wherein the at least one additional amino acidsubstitution occurs at EU position 356, 358, 431, 214 or a combinationthereof.
 41. The anti-SpA variant antibody of claim 40, wherein the IgG1allotype is a G1m17 allotype, and wherein the at least one amino acidsubstitution comprises an Arg at position 435, a Phe at position 436,and Gln at position 274 (SEQ ID NO: 40).
 42. An anti-SpA variantantibody comprising an anti-SpA Fc variant antibody derived from aparental anti-SpA antibody that is a human anti-SpA antibody that has avariable light chain sequence, a variable heavy chain sequence, or both,which further comprise one or more maturation mutations resulting in oneor more amino acid substitutions, deletions, or insertions that improveone or more properties of the anti-SpA variant antibody selected fromaffinity, avidity, stability, solubility, expression level, and/orbiological activity.
 43. An anti-Staphylococcus aureus (S. aureus)antibody comprising aparental antibody derived from an IGHV-3 familygerm line V gene sequence having one or more heavy chain variable domainamino acid substitutions, resulting in attenuation of non-immune, SpAsuper-antigen type binding to the heavy chain variable domain of theanti-S. aureus antibody as compared to the parental anti-S. aureusantibody.
 44. The anti-S. aureus variable heavy chain sequence variantantibody of claim 43, wherein the variable heavy chain sequence variantantibody is an anti-SpA antibody and wherein the heavy chain constantdomain comprises an amino acid sequence selected from SEQ ID NOs: 31-56.45. An anti-S. aureus variable heavy chain sequence variant antibodycomprising an immunoglobulin light chain and a variant immunoglobulinheavy chain which comprises a variable heavy chain sequence and aconstant heavy chain sequence, wherein the variant immunoglobulin heavychain comprises one or more amino acid substitutions in its variableheavy chain sequence as compared to a parental anti-S. aureus antibody,wherein the one or more amino acid substitutions include one or moreKabat positions selected from heavy chain positions H15, H17, H19, H57,H59, H64, H65, H66, H68, H69, H70, H80, H81 and, H82a, H82b.
 46. Theanti-S. aureus variable heavy chain sequence variant antibody of claim45, wherein the variable heavy chain sequence variant antibody is ananti-SpA antibody.
 47. The anti-S. aureus variable heavy chain sequencevariant antibody of claim 45, wherein the variable heavy chain sequencevariant antibody is an anti-SpA antibody and wherein the heavy chainconstant domain comprises an amino acid sequence selected from SEQ IDNOs: 31-56.
 48. The anti-S. aureus variable heavy chain sequence variantantibody of claim 45, wherein the parental antibody is an anti-SpAhumanized antibody, an anti-SpA Fc variant antibody, an anti-SpA maturedantibody, or an anti-SpA matured Fc variant antibody; wherein thevariable light chain sequence and/or the variable heavy chain sequenceof the anti-SpA matured antibody, or anti-SpA matured Fc variantantibody comprise one or more maturation mutations resulting in one ormore amino acid substitutions, deletions, or insertions that improve oneor more properties of the humanized anti-SpA antibody selected fromaffinity, avidity, stability, solubility, expression level, and/orbiological activity.
 49. The anti-S. aureus variable heavy chainsequence variant antibody of claim 48 comprising: an immunoglobulinheavy chain which comprises a variable heavy chain sequence and aconstant heavy chain sequence, wherein the variable heavy chain sequencecomprises (i) a heavy chain CDR1 sequence selected from GFAFSNYD,GFTFNTNA, and GYSFTSYY; (ii) a heavy chain CDR2 sequence selected fromISSGGTYP, IRSKSNNYAT, and IDPFNGGT; and (iii) a heavy chain CDR3sequence selected from ARGGFLITTRDYYAMDY, VTEHYDYDYYVMDY, andARYGYDGTFYAMDY; and an immunoglobulin light chain which comprises avariable light chain sequence and a constant light chain sequence,wherein the variable light chain sequence comprises (i) a light chainCDR1 sequence selected from SSVSY and ESVEYSGASL; (ii) a light chainCDR2 sequence selected from DTS, AAS, and EIS; and (iii) a light chainCDR3 sequence selected from QQWSSYPPT, QQSRKVPST, and QQWSYPFT.
 50. Theanti-SpA antibody of claim 45, wherein the one or more amino acidsubstitutions are selected from: H17S to H17P; H19R to H19G. H19K orH19T; H57K to H57A, H57P, H57R, or H57S; H57I to H57A, H57P, H57R, orH57S; H57T to H57A, H57P, H57R, or H57S; H59Y to H59F, H59H, H59N, orH59S; H68T to H68A, H68I or H68S; H70S to H70F; H81Q to H81E, H81H orH81R; H82aN to H82aD, H82aH, H82aK, H82aS, H82aT; H82aG to H82aD, H82aH,H82aK, H82aS, H82aT; and H82bS to H82bG, H82bN, or H82bT.